Search

Calorie restriction is associated with enhanced intestinal regeneration after irradiation, but the requirement of autophagy for this process is not known. Our data support the premise that intestinal epithelial autophagy is required for the regenerative benefit of calorie restriction. We also report that luminal levels of primary bile acid glycocholic acid are modulated by epithelial cell autophagy during calorie restriction with direct effects on epithelial stem cell function.

The profound complexity of the intestinal mucosa demands a spatial approach to the study of gut transcriptomics. Although single-cell RNA sequencing has revolutionized our ability to survey the diverse cell types of the intestine, knowledge of cell type alone cannot fully describe the cells that make up the intestinal mucosa. During homeostasis and disease, dramatic gradients of oxygen, nutrients, extracellular matrix proteins, morphogens, and microbiota collectively dictate intestinal cell state, and only spatial techniques can articulate differences in cellular transcriptomes at this level. Spatial transcriptomic techniques assign transcriptomic data to precise regions in a tissue of interest. In recent years, these protocols have become increasingly accessible, and their application in the intestinal mucosa has exploded in popularity. In the gut, spatial transcriptomics typically involve the application of tissue sections to spatially barcoded RNA sequencing or laser capture microdissection followed by RNA sequencing. In combination with single-cell RNA sequencing, these spatial sequencing approaches allow for the construction of spatial transcriptional maps at pseudosingle-cell resolution. In this review, we describe the spatial transcriptomic technologies recently applied in the gut and the previously unattainable discoveries that they have produced.

Colorectal cancer (CRC) is a leading cause of cancer-related death. There is an urgent need for new methods of early CRC detection and monitoring to improve patient outcomes. Extracellular vesicles (EVs) are secreted, lipid-bilayer bound, nanoparticles that carry biological cargo throughout the body and in turn exhibit cancer-related biomarker potential. RNA binding proteins (RBPs) are posttranscriptional regulators of gene expression that may provide a link between host cell gene expression and EV phenotypes. Insulin-like growth factor 2 RNA binding protein 1 (IGF2BP1/IMP1) is an RBP that is highly expressed in CRC with higher levels of expression correlating with poor prognosis. IMP1 binds and potently regulates tumor-associated transcripts that may impact CRC EV phenotypes. Our objective was to test whether IMP1 expression levels impact EV secretion and/or cargo. We used RNA sequencing, in vitro CRC cell lines, ex vivo colonoid models, and xenograft mice to test the hypothesis that IMP1 influences EV secretion and/or cargo in human CRC. Our data demonstrate that IMP1 modulates the RNA expression of transcripts associated with extracellular vesicle pathway regulation, but it has no effect on EV secretion levels in vitro or in vivo. Rather, IMP1 appears to affect EV regulation by directly entering EVs in a transformation-dependent manner. These findings suggest that IMP1 has the ability to shape EV cargo in human CRC, which could serve as a diagnostic/prognostic circulating tumor biomarker.

Colorectal cancer (CRC) is among the most frequent forms of cancer, and new strategies for its prevention and therapy are urgently needed

Intestinal epithelial transit amplifying cells are essential stem progenitors required for intestinal homeostasis, but their rapid proliferation renders them vulnerable to DNA damage from radiation and chemotherapy. Despite their critical roles in intestinal homeostasis and disease, few studies have described genes that are essential to transit amplifying cell function. We report that the RNA methyltransferase, METTL3, is required for survival of transit amplifying cells in the murine small intestine. Transit amplifying cell death after METTL3 deletion was associated with crypt and villus atrophy, loss of absorptive enterocytes, and uniform wasting and death in METTL3-depleted mice. Ribosome profiling and sequencing of methylated RNAs in enteroids andin vivodemonstrated decreased translation of hundreds of unique methylated transcripts after METTL3 deletion, particularly transcripts involved in growth factor signal transduction such asKras. Further investigation confirmed a novel relationship between METTL3 andKrasmethylation and protein levelsin vivo. Our study identifies METTL3 as an essential factor supporting the homeostasis of small intestinal tissue via direct maintenance of transit amplifying cell survival. We highlight the crucial role of RNA modifications in regulating growth factor signaling in the intestine, with important implications for both homeostatic tissue renewal and epithelial regeneration.

Supp. Fig. 1 legend

PDF file - 52K

Supplementary Figure 1. Dermo1Cre;Rosa-CAG-LSL-TdTomato mice.

PDF file - 193K

Purpose: BRAF inhibitors (BRAFi) extend survival in BRAF-mutant melanoma but can promote the growth of Ras-mutant neoplasms. This study determined if gastrointestinal polyps found in BRAFi-treated patients harbored Ras mutations.Experimental Design: Colonic and gastric polyps were identified and resected from BRAFi-treated melanoma patients. Next-generation sequencing (NGS) was performed on polyps. The ability of BRAFi to promote polyp formation was functionally characterized in Apc Min+/− mice. MAPK and β-catenin pathway activity was assessed by immunohistochemistry in mouse and human polyps.Results: Fourteen patients treated with BRAFi underwent endoscopy to assess for polyps. Seven out of 7 patients >40 years of age and treated for >2 years were found to have colonic tubular adenomas with 4 out of the 7 patients having 5 or more polyps. One patient presented with bleeding from hyperplastic gastric polyps that recurred 6 months after BRAFi rechallenge. NGS performed on polyps found no mutations in MAPK pathway genes, but found APC mutations in all tubular adenomas. A significant increase in the number of polyps was observed in BRAFi-treated compared with control-treated Apc Min+/− mice (20.8 ± 9.2 vs 12.8 ± 0.1; P = 0.016). No polyps were observed in BRAFi-treated wild-type mice.Conclusions: BRAFi may increase the risk of developing hyperplastic gastric polyps and colonic adenomatous polyps. Due to the risk of gastrointestinal bleeding and the possibility of malignant transformation, further studies are needed to determine whether or not endoscopic surveillance should be recommended for patients treated with BRAFi. Clin Cancer Res; 21(23); 5215–21. ©2015 AACR.

Supplemental Figure 1. Endoscopic and histological H&E images of colonic polyps in a Magnification: 10X (bottom left); 200X (bottom right) BRAF mutant melanoma patient treated with long-term vemurafenib.

Supplemental Methods from Multiple Gastrointestinal Polyps in Patients Treated with BRAF Inhibitors

Understanding how macroautophagy/autophagy contributes to tissue homeostasis is essential for understanding organismal health. The intestinal epithelium is an ideal model to define mechanisms that regulate tissue homeostasis because it houses well-defined populations of intestinal stem cells. Active intestinal stem cells (a-ISCs) are defined by their active cycling and self-renewal during homeostasis, which supports continual tissue turnover in vivo. In vitro, this is observed as long-term organoid formation capacity. A second population of stem cells, called “facultative intestinal stem cells” (f-ISCs), are defined by their ability to 1) survive tissue damage that depletes the injury-sensitive a-ISCs and 2) reenter the cell cycle to repopulate the a-ISC compartment and regenerate the epithelium. The prospective identification of f-ISCs has been challenging, as cells expressing markers of multiple differentiated lineages, particularly secretory lineages, appear to function as f-ISCs in diverse injury contexts. We evaluated cell age (defined as time elapsed after cell cycle exit) and autophagic state (marked by autophagic vesicle content) as molecular features that may be related to f-ISC capacity. We found that autophagic state, but not cell age, prospectively identifies f-ISCs within multiple lineages. As such, we describe autophagy as a lineage-agnostic marker of f-ISC capacity in the mammalian intestine.

3D patient-derived organoids (PDOs) have been utilized to evaluate potential therapies for patients with different cancers. However, the use of PDOs created from treatment-naive patient biopsies for prediction of clinical outcomes in patients with esophageal cancer has not yet been reported. Herein we describe a pilot prospective observational study with the goal of determining whether esophageal cancer PDOs created from treatment naive patients can model or predict clinical outcomes. Endoscopic biopsies of treatment-naive patients at a single tertiary care center were used to generate esophageal cancer PDOs, which were treated with standard-of-care chemotherapy, gamma-irradiation, and newer non-standard approaches, such as proton beam therapy or two small molecule inhibitors. Clinical outcomes of patients following neoadjuvant treatment were compared to their in vitro PDO responses, demonstrating the PDO’s ability to mirror clinical response, suggesting the value of PDOs in prediction of clinical response to new therapeutic approaches. Future prospective clinical trials should test the use of pre-treatment PDOs to identify specific, targeted therapies for individual patients with esophageal adenocarcinoma.

The intestinal epithelium exhibits a rapid and efficient regenerative response to injury. Emerging evidence supports a model where plasticity of differentiated cells, particularly those in the secretory lineages, contributes to epithelial regeneration upon ablation of injury-sensitive stem cells. However, such facultative stem cell activity is rare within secretory populations. Here, we ask whether specific functional properties predict facultative stem cell activity. We utilize in vivo labeling combined with ex vivo organoid formation assays to evaluate how cell age and autophagic state contribute to facultative stem cell activity within secretory lineages. Strikingly, we find that cell age (time elapsed since cell cycle exit) does not correlate with secretory cell plasticity. Instead, high autophagic vesicle content predicts plasticity and resistance to DNA damaging injury independently of cell lineage. Our findings indicate that autophagic status prior to injury serves as a lineage-agnostic marker for the prospective identification of facultative stem cells.

Small-molecule inhibitors of enzyme function are critical tools for the study of cell biological processes and for treatment of human disease. Identifying inhibitors with suitable specificity and selectivity for single enzymes, however, remains a challenge. In this study we describe our serendipitous discovery that NMS-873, a compound that was previously identified as a highly selective allosteric inhibitor of the ATPase valosin-containing protein (VCP/p97), rapidly induces aerobic fermentation in cultured human and mouse cells. Our further investigation uncovered an unexpected off-target effect of NMS-873 on mitochondrial oxidative phosphorylation, specifically as a dual inhibitor of Complex I and ATP synthase. This work points to the need for caution regarding the interpretation of cell survival data associated with NMS-873 treatment and indicates that cellular toxicity associated with its use may be caused by both VCP/p97-dependent and VCP/p97-independent mechanisms.

Phagocytosis is the necessary first step to sense foreign microbes or particles and enables activation of innate immune pathways such as inflammasomes. However, the molecular mechanisms underlying how phagosomes modulate inflammasome activity are not fully understood. We show that in murine dendritic cells (DCs), the lysosomal histidine/peptide solute carrier transporter SLC15A4, associated with human inflammatory disorders, is recruited to phagosomes and is required for optimal inflammasome activity after infectious or sterile stimuli. Dextran sodium sulfate-treated SLC15A4-deficient mice exhibit decreased colon inflammation, reduced IL-1β production by intestinal DCs, and increased autophagy. Similarly, SLC15A4-deficient DCs infected with Salmonella typhimurium show reduced caspase-1 cleavage and IL-1β production. This correlates with peripheral NLRC4 inflammasome assembly and increased autophagy. Overexpression of constitutively active mTORC1 rescues decreased IL-1β levels and caspase1 cleavage, and restores perinuclear inflammasome positioning. Our findings support that SLC15A4 couples phagocytosis with inflammasome perinuclear assembly and inhibition of autophagy through phagosomal content sensing. Our data also reveal the previously unappreciated importance of mTORC1 signaling pathways to promote and sustain inflammasome activity.

The facilitative glucose transport GLUT1 (SLC2A1) is a constitutively expressed membrane protein involved in basal uptake of blood glucose. GLUT1 modification by N-linked glycosylation at a single asparagine residue (N45) appears to play multiple roles in the trafficking, stability and transport activity of this protein. Here we examine the role of complex N-glycosylation on GLUT1 function in renal epithelial cells by arresting this modification at the high-mannose stage with the mannosidase I inhibitor kifunensine. Consistent with prior work in which GLUT1 glycosylation was completely inhibited, we find that kifunensine treatment results in a time-dependent decrease of up to 40% in cellular glucose uptake. We further demonstrate that this effect is primarily a result of deficient GLUT1 trafficking to the cell membrane due to quality control mechanisms that instead direct GLUT1 to the ER-associated degradation (ERAD) pathway. Unlike tunicamycin, which inhibits the first step in N-glycosyl transfer and causes dramatic cell cycle arrest, kifunensine causes only a modest decrease in GLUT1 levels and cell cycle progression in both normal and transformed renal cells. The effect of kifunensine on the cell cycle appears to be independent of its effect on GLUT1, since all renal cell types in this study displayed decreased proliferation regardless of their dependence on glucose uptake for growth and survival. Together these results indicate that proper N-glycan processing plays an important role in directing GLUT1 to the cell surface and that disruption of mannosidase activity results in aberrant degradation of GLUT1 by the ERAD pathway.

Phagocytosis is the first step in the assessment of foreign microbes or particles and enables activation of innate immune pathways such as the inflammasome. However, missing links between phagosomes and inflammasomes remain to be discovered. We show that in murine dendritic cells (DCs) the lysosomal histidine/peptide solute carrier transporter SLC15A4, associated with human inflammatory disorders, is recruited to phagosomes and is required for optimal inflammasome activity after infectious or sterile stimuli. Dextran sodium sulphate-treated SLC15A4-deficient mice exhibit decreased colon inflammation, reduced IL-1β production by intestinal DCs and increased autophagy. Similarly, SLC15A4-deficient DCs infected withSalmonellatyphimurium show reduced caspase-1 cleavage and IL-1β production. This correlates with peripheral NLRC4 inflammasome assembly and increased autophagy. Overexpression of constitutively active mTORC1 rescues decreased IL-1β levels and caspase-1 cleavage, and restores perinuclear inflammasome positioning. Our findings suggest that SLC15A4 is a novel link that couples phagocytosis with inflammasome perinuclear assembly and inhibition of autophagy through phagosomal content sensing. Our data also reveal the previously unappreciated importance of mTORC1 signaling pathways to promote and sustain inflammasome activity.

Homeostatic tissue maintenance requires coordinated regulation of metabolic processes including macroautophagy/autophagy. Autophagy dysregulation underlies numerous human diseases. Our prior work revealed that the RNA binding protein IGF2BP1/IMP1 binds transcripts encoding autophagy-related proteins. Furthermore, Imp1 deletion in gastrointestinal epithelial cells in mice was associated with enhanced autophagy flux and improved recovery from tissue injury. In the current study, we evaluated molecular mechanisms underlying IMP1 modulation of autophagy. We provide a mechanism of direct IMP1 regulation of MAP1LC3B that is dependent upon IMP1 phosphorylation or cell stress, suggesting dynamic modulation of Imp1-mediated autophagy repression that facilitates tissue regeneration. More broadly, our study supports a new mechanism by which tissue regeneration is modulated post-transcriptionally via cell state rather than changes in stem or other cell lineages. This new mechanism may be particularly important in gastrointestinal epithelial cells, where autophagy is essential for tissue recovery following injury, or in diseases such as inflammatory bowel disease where defective autophagy is implicated.

Here, we introduce a facile, scalable engineering approach to enable long-term development and maturation of organoids. We have redesigned the configuration of conventional organoid culture to develop a platform that converts single injections of stem cell suspensions to radial arrays of organoids that can be maintained for extended periods without the need for passaging. Using this system, we demonstrate accelerated production of intestinal organoids with significantly enhanced structural and functional maturity, and their continuous development for over 4 weeks. Furthermore, we present a patient-derived organoid model of inflammatory bowel disease (IBD) and its interrogation using single-cell RNA sequencing to demonstrate its ability to reproduce key pathological features of IBD. Finally, we describe the extension of our approach to engineer vascularized, perfusable human enteroids, which can be used to model innate immune responses in IBD. This work provides an immediately deployable platform technology toward engineering more realistic organ-like structures in a dish.

The classical methods for determining glucose uptake rates in living cells involve the use of isotopically labeled 2-deoxy- d -glucose or 3-O-methyl- d -glucose, which enter cells via well-characterized membrane transporters of the SLC2A and SLC5A families, respectively. These classical methods, however, are increasingly being displaced by high-throughput assays that utilize fluorescent analogs of glucose. Among the most commonly used of these analogs are 2-NBDG and 6-NBDG, which contain a bulky 7-nitro-2,1,3-benzoxadiazol-4-yl-amino moiety in place of a hydroxy group on d -glucose. This fluorescent group significantly alters both the size and shape of these molecules compared to glucose, calling into question whether they actually enter cells by the same transport mechanisms. In this study, we took advantage of the well-defined glucose uptake mechanism of L929 murine fibroblasts, which rely exclusively on the Glut1/Slc2a1 membrane transporter. We demonstrate that neither pharmacologic inhibition of Glut1 nor genetic manipulation of its expression has a significant impact on the binding or uptake of 2-NBDG or 6-NBDG by L929 cells, though both approaches significantly impact [3H]-2-deoxyglucose uptake rates. Together these data indicate that 2-NBDG and 6-NBDG can bind and enter mammalian cells by transporter-independent mechanisms, which calls into question their utility as an accurate proxy for glucose transport.

Although basal cell hyperplasia is a histologic hallmark of eosinophilic esophagitis (EoE), little is known about the capabilities of epithelial renewal and differentiation in the EoE inflammatory milieu. In murine esophageal epithelium, there are self-renewing and slowly proliferating basal stem-like cells characterized by concurrent expression of CD73 (5'-nucleotidase ecto) and CD104 (integrin β4). Here, we investigated CD73We performed flow cytometry on esophageal biopsy samples from EoE and non-EoE patients to determine the quantity of CD73EoE patients showed decreased epithelial CD73This study addressed the role of CD73

This protocol describes dissociation of a human intestinal biopsy tissue into single cells, followed by depletion of dead cells via annexin V MACS beads. The outcome is a single-cell suspension with viability ≥ 90% that is used for single-cell sequencing or establishemt of enteroid/colonoid culture.

Stratified squamous epithelium of the esophagus is comprised of basal keratinocytes that execute a terminal differentiation program in overlying suprabasal and superficial cell layers. Although morphologic progression coupled with expression of specific molecular markers has been characterized along the esophageal epithelial differentiation gradient, the molecular heterogeneity within the cell types along this trajectory has yet to be classified at the level of single cell resolution. To explore the molecular characteristics of esophageal keratinocytes along the squamous differentiation continuum, we performed single cell RNA-Sequencing transcriptomic profiling of 7,972 cells from murine esophageal epithelial sheets. We identified 8 distinct cell clusters in esophageal epithelium, unveiling an unexpected level of diversity, particularly among basal cells. We further mapped the cellular pathways and lineage trajectories within basal, suprabasal, and superficial clusters as well as within the heterogeneous basal cell populations, providing a comprehensive molecular view of esophageal epithelial cells in the context of squamous differentiation. Finally, we explored the impact of tissue aging upon esophageal epithelial cell clusters and demonstrated that mitochondrial dysfunction is a feature of aging in normal esophageal epithelium. These studies provide an unparalleled molecular perspective on murine esophageal keratinocytes that will serve as a valuable resource for dissecting cell type-specific roles in esophageal biology under conditions of homeostasis, aging, and tissue pathology.

Although morphologic progression coupled with expression of specific molecular markers has been characterized along the esophageal squamous differentiation gradient, the molecular heterogeneity within cell types along this trajectory has yet to be classified at the single cell level. To address this knowledge gap, we perform single cell RNA-sequencing of 44,679 murine esophageal epithelial, to identify 11 distinct cell populations as well as pathways alterations along the basal-superficial axis and in each individual population. We evaluate the impact of aging upon esophageal epithelial cell populations and demonstrate age-associated mitochondrial dysfunction. We compare single cell transcriptomic profiles in 3D murine organoids and human esophageal biopsies with that of murine esophageal epithelium. Finally, we employ pseudotemporal trajectory analysis to develop a working model of cell fate determination in murine esophageal epithelium. These studies provide comprehensive molecular perspective on the cellular heterogeneity of murine esophageal epithelium in the context of homeostasis and aging.

Summary: Juvenile Polyposis Syndrome is a pediatric cancer predisposition syndrome for which approximately half of patients do not have a known germline driver; we propose BMPR2 as a possible germline driver of disease in mutation-negative JPS. Background: Juvenile Polyposis Syndrome (JPS) is a cancer predisposition syndrome characterized in some cases by inactivating germline mutations in BMPR1A or SMAD4. However, in 40-60% of patients the germline driver is unknown. Methods: Through whole exome sequencing, an individual with high polyp burden and negative genetic testing for variants in SMAD4 and BMPR1A was found to have a potentially inactivating germline variant in BMPR2. Under an IRB-approved protocol, patient-derived, three-dimensional organoids were established from adjacent colon and polyp tissue of this individual, as well as an individual with a BMPR1A deletion and age- and sex-matched controls. Proliferation, metabolic activity, and protein expression of each line were measured by Western blot, Ki67 staining, EdU labelling, cell titer glo (CTG) assay. Results: Distinct phenotypic differences are evident between colonoids from an individual with a BMPR1A deletion and both the colon and polyp BMPR2 variant colonoids, such as increased crypt budding and Ki67 staining. CTG assay data indicate higher metabolic activity in BMPR2 variant colonoids in comparison to normal controls (p= 0.0104.) Through Western blot analysis, both BMPR2 colon and polyp colonoids showed decreased SMAD4 expression and phosphorylation. Conclusions: These data demonstrate that clear phenotypic differences exist between colonoid lines established from normal controls, individuals with known JPS predisposition genes, and a novel candidate driver of disease, BMPR2. Proliferation and protein expression data suggest that both known and candidate genotypes are consistent with a hyperproliferative phenotype. In vitro data support BMPR2 as a candidate germline driver of the JPS phenotype, and additional study is ongoing regarding downstream pathway regulation in colonoids with a BMPR2 variant. Citation Format: Suzanne P. MacFarland, Bridgid Greed, Michael H. Xie, Garrett M. Brodeur, Zvi Cramer, Maiah H. Dent, Melani M. Duvall, Kathryn E. Hamilton, James Howe, Tatiana Karakasheva, Petar Mamula, Christopher Lengner. Bone morphogenic protein receptor 2 (BMPR2) as a potential germline driver in Juvenile Polyposis Syndrome (JPS) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5898.

As the interface between the gut microbiota and the mucosal immune system, there has been great interest in the maintenance of colonic epithelial integrity through mitochondrial oxidation of butyrate, a short-chain fatty acid produced by the gut microbiota. Herein, we showed that the intestinal epithelium could also oxidize long-chain fatty acids, and that luminally delivered acylcarnitines in bile could be consumed via apical absorption by the intestinal epithelium, resulting in mitochondrial oxidation. Finally, intestinal inflammation led to mitochondrial dysfunction in the apical domain of the surface epithelium that may reduce the consumption of fatty acids, contributing to higher concentrations of fecal acylcarnitines in murine Citrobacter rodentium-induced colitis and human inflammatory bowel disease. These results emphasized the importance of both the gut microbiota and the liver in the delivery of energy substrates for mitochondrial metabolism by the intestinal epithelium.

Intestinal epithelial cells are among the most rapidly proliferating cell types in the human body. There are several different subtypes of epithelial cells, each with unique functional roles in responding to the ever-changing environment. The epithelium’s ability for rapid and customized responses to environmental changes requires multitiered levels of gene regulation. An emerging paradigm in gastrointestinal epithelial cells is the regulation of functionally related mRNA families, or regulons, via RNA-binding proteins (RBPs). RBPs represent a rapid and efficient mechanism to regulate gene expression and cell function. In this review, we will provide an overview of intestinal epithelial RBPs and how they contribute specifically to intestinal epithelial stem cell dynamics. In addition, we will highlight key gaps in knowledge in the global understanding of RBPs in gastrointestinal physiology as an opportunity for future studies.

The homeostatic proliferation-differentiation gradient in the esophageal epithelium is perturbed under inflammatory disease conditions such as gastroesophageal reflux disease and eosinophilic esophagitis. Herein we describe the protocols for rapid generation (

Esophageal squamous cell carcinoma (ESCC) is amongst the most aggressive human malignancies, representing a significant health burden worldwide. Autophagy is an evolutionarily conserved catabolic process that degrades and recycles damaged organelles and misfolded proteins to maintain cellular homeostasis. Alterations in autophagy are associated with cancer pathogenesis, including ESCC; however, the functional role of autophagy in ESCC remains elusive. Here, we discuss the clinical relevance of autophagy effectors in ESCC and review current knowledge regarding the molecular mechanisms through which autophagy contributes to ESCC. We highlight the cytoprotective role of autophagy in ESCC and discuss autophagy inhibitors as novel experimental therapeutics to potentiate the effects of anti-cancer therapies and/or to overcome therapeutic resistance in ESCC.

Summary Two principal stem cell pools orchestrate the rapid cell turnover in the intestinal epithelium. Rapidly cycling Lgr5+ stem cells are intercalated between the Paneth cells at the crypt base (CBCs) and injury-resistant reserve stem cells reside above the crypt base. The intermediate filament Keratin 15 (Krt15) marks either stem cells or long-lived progenitor cells that contribute to tissue repair in the hair follicle or the esophageal epithelium. Herein, we demonstrate that Krt15 labels long-lived and multipotent cells in the small intestinal crypt by lineage tracing. Krt15+ crypt cells display self-renewal potential in vivo and in 3D organoid cultures. Krt15+ crypt cells are resistant to high-dose radiation and contribute to epithelial regeneration following injury. Notably, loss of the tumor suppressor Apc in Krt15+ cells leads to adenoma and adenocarcinoma formation. These results indicate that Krt15 marks long-lived, multipotent, and injury-resistant crypt cells that may function as a cell of origin in intestinal cancer., Highlights • Krt15 marks multipotent and self-renewing crypt cells in the mouse small intestine • Krt15+ crypt cells are radio-resistant and contribute to regeneration following injury • Apc loss in Krt15+ cells leads to intestinal adenoma and adenocarcinoma formation • Krt15+ cells may function as a cell of origin in intestinal cancer, In this article, Rustgi and colleagues identify a new population of long-lived cells within the intestinal crypt, marked by the Krt15 promoter. Krt15+ cells are self-renewing, multipotent, and radio-resistant cells that facilitate recovery from radiation-induced injury. Furthermore, such cells, when targeted with Apc loss, undergo transformation to adenomas and adenocarcinomas.

The RNA-binding protein LIN28B plays an important role in development, stem cell biology, and tumorigenesis. LIN28B has two isoforms: the LIN28B-long and -short isoforms. Although studies have revealed the functions of the LIN28B-long isoform in tumorigenesis, the role of the LIN28B-short isoform remains unclear and represents a major gap in the field. The LIN28B-long and -short isoforms are expressed in a subset of human colorectal cancers and adjacent normal colonic mucosa, respectively. To elucidate the functional and mechanistic aspects of these isoforms, colorectal cancer cells (Caco-2 and LoVo) were generated to either express no LIN28B or the -short or -long isoform. Interestingly, the long isoform suppressed LET-7 expression and activated canonical RAS/ERK signaling, whereas the short isoform did not. The LIN28B-long isoform–expressing cells demonstrated increased drug resistance to 5-fluorouracil and cisplatin through the upregulation of ERCC1, a DNA repair gene, in a LET-7–dependent manner. The LIN28B-short isoform preserved its ability to bind pre-let-7, without inhibiting the maturation of LET-7, and competed with the LIN28B-long isoform for binding to pre-let-7. Coexpression of the short isoform in the LIN28B-long isoform–expressing cells rescued the phenotypes induced by the LIN28B-long isoform. Implications: This study demonstrates the differential antagonistic functions of the LIN28B-short isoform against the LIN28B-long isoform through an inability to degrade LET-7, which leads to the novel premise that the short isoform may serve to counterbalance the long isoform during normal colonic epithelial homeostasis, but its downregulation during colonic carcinogenesis may reveal the protumorigenic effects of the long isoform. Mol Cancer Res; 16(3); 403–16. ©2018 AACR.

Colorectal cancer (CRC) is the fourth most common cancer in both men and women in the United States, resulting in over 55,000 deaths annually. Environmental and genetic factors influence the development of CRC, and inflammation is a critical hallmark of cancer that may arise from a variety of factors.While patients with inflammatory bowel disease (IBD) have a higher risk of developing CRC, sporadic CRCs may engender or be potentiated by inflammation as well. In this review, we focus on recent advances in basic and translational research utilizing murine models to understand the contribution of inflammatory signaling pathways to CRC.We discuss advances in the utility of three-dimensional enteroid/colonoid/tumoroid cultures to understand immune-epithelial interactions in CRC, as well as the potential for utilizing patient-derived tumoroids for personalized therapies.This review underscores the importance of understanding the complex molecular mechanisms underlying inflammation in sporadic CRC and highlights up-and-coming or new avenues for CRC biomarkers or therapies.

Paneth cells (PCs), a secretory population located at the base of the intestinal crypt, support the intestinal stem cells (ISC) with growth factors and participate in innate immunity by releasing antimicrobial peptides, including lysozyme and defensins. PC dysfunction is associated with disorders such as Crohn's disease and necrotizing enterocolitis, but the specific pathways regulating PC development and function are not fully understood. Here we tested the role of the neuregulin receptor ErbB3 in control of PC differentiation and the ISC niche. Intestinal epithelial ErbB3 knockout caused precocious appearance of PCs as early as postnatal day 7, and substantially increased the number of mature PCs in adult mouse ileum. ErbB3 loss had no effect on other secretory lineages, but increased expression of the ISC marker Lgr5. ErbB3-null intestines had elevated levels of the Atoh1 transcription factor, which is required for secretory fate determination, while Atoh1+ cells had reduced ErbB3, suggesting reciprocal negative regulation. ErbB3-null intestinal progenitor cells showed reduced activation of the PI3K–Akt and ERK MAPK pathways. Inhibiting these pathways in HT29 cells increased levels of ATOH1 and the PC marker LYZ. Conversely, ErbB3 activation suppressed LYZ and ATOH1 in a PI3K-dependent manner. Expansion of the PC compartment in ErbB3-null intestines was accompanied with elevated ER stress and inflammation markers, raising the possibility that negative regulation of PCs by ErbB3 is necessary to maintain homeostasis. Taken together, our data suggest that ErbB3 restricts PC numbers through PI3K-mediated suppression of Atoh1 levels leading to inhibition of PC differentiation, with important implications for regulation of the ISC niche.

RNA binding proteins, including IMP1/IGF2BP1, are essential regulators of intestinal development and cancer. Imp1 hypomorphic mice exhibit gastrointestinal growth defects, yet the specific role for IMP1 in colon epithelial repair is unclear. Our prior work revealed that intestinal epithelial cell‐specific Imp1 deletion ( Imp1 Δ IEC ) was associated with better regeneration in mice after irradiation. Here, we report increased IMP1 expression in patients with Crohn9s disease and ulcerative colitis. We demonstrate that Imp1 Δ IEC mice exhibit enhanced recovery following dextran sodium sulfate (DSS)‐mediated colonic injury. Imp1 Δ IEC mice exhibit Paneth cell granule changes, increased autophagy flux, and upregulation of Atg5. In silico and biochemical analyses revealed direct binding of IMP1 to MAP1LC3B , ATG3, and ATG5 transcripts. Genetic deletion of essential autophagy gene Atg7 in Imp1 Δ IEC mice revealed increased sensitivity of double‐mutant mice to colonic injury compared to control or Atg7 single mutant mice, suggesting a compensatory relationship between Imp1 and the autophagy pathway. The present study defines a novel interplay between IMP1 and autophagy, where IMP1 may be transiently induced during damage to modulate colonic epithelial cell responses to damage.