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Salmonella enterica serovar Typhimurium (STm) is a causative pathogen for robust inflammatory gastrointestinal disease and can lead to systemic infection. Eicosanoids, bioactive lipid mediators, play a crucial role in modulating both the induction and resolution of inflammatory responses during an infection. A subset of eicosanoids activates PPARs, nuclear receptor/transcription factors that regulate fatty acid metabolism, lipid body formation, and macrophage function. In this study, we determined that mice lacking PPARα exhibited reduced inflammatory hallmarks of STm infection, including lower inflammatory gene expression, cecal inflammation, and bacterial dissemination, along with a significant increase in cecal eicosanoid metabolism compared to wildtype C57BL/6 mice. In macrophages, STm favored M2b-polarized macrophages for intracellular infection, leading to reduced arachidonic acid and ceramide production. Inhibition of fatty acid oxidation via Etomoxir in STm-infected macrophages reduced bacterial burdens and promoted cell death. In Etomoxir-treated wildtype mice, STm infection increased ceramide production, decreased inflammatory gene expression in the cecum, and increased the number of STm-containing M1 macrophages in mesenteric lymph nodes. These findings revealed a novel role for the lipid-immune signaling axis in Salmonella infections, providing significant insights into the lipid-mediated regulation of inflammation during bacterial infections in the gut.

Salmonella enterica serovar Typhimurium (STm) causes gastroenteritis and can progress to reactive arthritis (ReA). STm forms biofilms in the gut that secrete the amyloid curli, which we previously demonstrated can trigger autoimmunity in mice. HLA-B27 is a genetic risk factor for ReA; activation of the unfolded protein response (UPR) due to HLA-B27 misfolding is thought to play a critical role in ReA pathogenesis. To determine whether curli exacerbates HLA-B27-induced UPR, bone marrow-derived macrophages (BMDMs) isolated from HLA-B27 transgenic (tg) mice were used. BMDMs treated with purified curli exhibited elevated UPR compared to C57BL/6, and curli-induced IL-6 was reduced by pre-treating macrophages with inhibitors of the IRE1α branch of the UPR. In BMDMs, intracellular curli colocalized with GRP78, a regulator of the UPR. In vivo, acute infection with wild-type STm increased UPR markers in the ceca of HLA-B27tg mice compared to C57BL/6. STm biofilms that contain curli were visible in the lumen of cecal tissue sections. Furthermore, curli was associated with macrophages in the lamina propria, colocalizing with GRP78. Together, these results suggest that UPR plays a role in the curli-induced inflammatory response, especially in the presence of HLA-B27, a possible mechanistic link between STm infection and genetic susceptibility to ReA.

Background & Aims: Autophagy plays roles in esophageal pathologies both benign and malignant. Here, we aim to define the role of autophagy in esophageal epithelial homeostasis. Methods: We generated tamoxifen-inducible, squamous epithelial-specific Atg7 (autophagy related 7) conditional knockout mice to evaluate effects on esophageal homeostasis and response to the carcinogen 4-nitroquinoline 1-oxide (4NQO) using histologic and biochemical analyses. We fluorescence-activated cell sorted esophageal basal cells based on fluorescence of the autophagic vesicle (AV)-identifying dye Cyto-ID and then subjected these cells to transmission electron microscopy, image flow cytometry, three-dimensional organoid assays, RNA sequencing, and cell cycle analysis. Three-dimensional organoids were subjected to passaging, single-cell RNA sequencing, cell cycle analysis, and immunostaining. Results: Genetic autophagy inhibition in squamous epithelium resulted in increased proliferation of esophageal basal cells under homeostatic conditions and also was associated with significant weight loss in mice treated with 4NQO that further displayed perturbed epithelial tissue architecture. Esophageal basal cells with high AV level (Cyto-IDHigh) displayed limited organoid formation capability on initial plating but passaged more efficiently than their counterparts with low AV level (Cyto-IDLow). RNA sequencing suggested increased autophagy in Cyto-IDHigh esophageal basal cells along with decreased cell cycle progression, the latter of which was confirmed by cell cycle analysis. Single-cell RNA sequencing of three-dimensional organoids generated by Cyto-IDLow and Cyto-IDHigh cells identified expansion of 3 cell populations and enrichment of G2/M-associated genes in the Cyto-IDHigh group. Ki67 expression was also increased in organoids generated by Cyto-IDHigh cells, including in basal cells localized beyond the outermost cell layer. Conclusions: Autophagy contributes to maintenance of the esophageal proliferation-differentiation gradient. Esophageal basal cells with high AV level exhibit limited proliferation and generate three-dimensional organoids with enhanced self-renewal capacity.

Background: Esophageal organoids from a variety of pathologies including cancer are grown in Advanced Dulbecco’s Modified Eagle Medium-Nutrient Mixture F12 (hereafter ADF). However, the currently available ADF-based formulations are suboptimal for normal human esophageal organoids, limiting the ability to compare normal esophageal organoids with those representing a given disease state. Methods: We have utilized immortalized normal human esophageal epithelial cell (keratinocyte) lines EPC1 and EPC2 and endoscopic normal esophageal biopsies to generate three-dimensional (3D) organoids. To optimize the ADF-based medium, we evaluated the requirement of exogenous epidermal growth factor (EGF) and inhibition of transforming growth factor-(TGF)-β receptor-mediated signaling, both key regulators of the proliferation of human esophageal keratinocytes. We have modeled human esophageal epithelial pathology by stimulating esophageal 3D organoids with interleukin (IL)-13, an inflammatory cytokine, or UAB30, a novel pharmacological activator of retinoic acid signaling. Results: The formation of normal human esophageal 3D organoids was limited by excessive EGF and intrinsic TGFβ-receptor-mediated signaling. Optimized HOME0 improved normal human esophageal organoid formation. In the HOME0-grown organoids, IL-13 and UAB30 induced epithelial changes reminiscent of basal cell hyperplasia, a common histopathologic feature in broad esophageal disease conditions including eosinophilic esophagitis. Conclusions: HOME0 allows modeling of the homeostatic differentiation gradient and perturbation of the human esophageal epithelium while permitting a comparison of organoids from mice and other organs grown in ADF-based media.

Barrett’s esophagus (BE) is a common precancerous lesion that can progress to esophageal adenocarcinoma (EAC). There are significant alterations in the esophageal microbiome in the progression from healthy esophagus to BE to EAC, including an increased abundance of a variety of lactate-producing bacteria and an increase of lactate in the tumor microenvironment, as predicted by metabolic modeling. The role of bacterial lactate in EAC is unknown. Here, we utilize patient-derived organoid (PDO) models of EAC and demonstrate that lactate inhibits the growth and proliferation of EAC PDOs through alterations in the tumor NADH/NAD+ redox state. Further RNA sequencing of EAC PDOs identifies ID1 and RSAD2 as potential regulatory molecules crucial in mediating lactate’s ability to suppress glycolysis and proliferation. Gene ontology analysis also identifies the activation of inflammatory and immunological pathways in addition to alterations in the metabolic pathways in EAC PDOs exposed to lactate, suggesting a multi-faceted role for lactate in the pathogenesis of EAC.

ABSTRACTThe Salmonella biofilm-associated amyloid protein, curli, is a dominant instigator of systemic inflammation and autoimmune responses following Salmonella infection. Systemic curli injections or infection of mice with Salmonella Typhimurium induce the major features of reactive arthritis, an autoimmune disorder associated with Salmonella infection in humans. In this study, we investigated the link between inflammation and microbiota in exacerbating autoimmunity. We studied C57BL/6 mice from two sources, Taconic Farms and Jackson Labs. Mice from Taconic Farms have been reported to have higher basal levels of the inflammatory cytokine IL − 17 than do mice from Jackson Labs due to the differences in their microbiota. When we systemically injected mice with purified curli, we observed a significant increase in diversity in the microbiota of Jackson Labs mice but not in that of the Taconic mice. In Jackson Labs, mice, the most striking effect was the expansion of Prevotellaceae. Furthermore, there were increases in the relative abundance of the family Akkermansiaceae and decreases in families Clostridiaceae and Muribaculaceae in Jackson Labs mice. Curli treatment led to significantly aggravated immune responses in the Taconic mice compared to Jackson Labs counterparts. Expression and production of IL − 1β, a cytokine known to promote IL − 17 production, as well as expression of Tnfa increased in the gut mucosa of Taconic mice in the first 24 hours after curli injections, which correlated with significant increases in the number of neutrophils and macrophages in the mesenteric lymph nodes. A significant increase in the expression of Ccl3 in colon and cecum of Taconic mice injected with curli was detected. Taconic mice injected with curli also had elevated levels of inflammation in their knees. Overall, our data suggest that autoimmune responses to bacterial ligands, such as curli, are amplified in individuals with a microbiome that promote inflammation.

Cytoplasmic accumulation of RNA binding protein, hnRNPK in cancer cells is associated with poor prognosis. Here the authors show that SCFFbxo4 E3 ubiquitin ligase-mediated polyubiquitylation of hnRNPK restricts c-Myc translation and limits cancer progression.

IntroductionUnder homeostatic conditions, esophageal epithelium displays a proliferation/differentiation gradient that is generated as proliferative basal cells give rise to suprabasal cells then terminally differentiated superficial cells. This proliferation/differentiation gradient is often perturbed in esophageal pathologies. Basal cell hyperplasia may occur in patients with gastroesophageal reflux disease (GERD), a condition in which acid from the stomach enters the esophagus, or eosinophilic esophagitis (EoE), an emerging form of food allergy. While GERD is a primary risk factor for esophageal cancer, epidemiological data suggests that EoE patients do not develop esophageal cancer.MethodsIn order to investigate the impact of EoE and esophageal cancer specifically on the cellular landscape of esophageal epithelium, we perform single cell RNA-sequencing in murine models of EoE and esophageal cancer, specifically esophageal squamous cell carcinoma (ESCC). We further evaluate modules of co-expressed genes in EoE- and ESCC-enriched epithelial cell clusters. Finally, we pair EoE and ESCC murine models to examine the functional relationship between these pathologies.ResultsIn mice with either EoE or ESCC, we find expansion of cell populations as compared to normal esophageal epithelium. In mice with EoE, we detect distinct expansion of 4 suprabasal populations coupled with depletion of 2 basal populations. By contrast, mice with ESCC display unique expansion of 2 basal populations and 1 suprabasal population, as well as depletion of 2 suprabasal populations. Senescence, glucocorticoid receptor signaling, and granulocyte-macrophage colony-stimulating factor pathways are associated with EoE-enriched clusters while pathways associated with cell proliferation and metabolism are identified in ESCC-enriched clusters. Finally, our in vivo data demonstrate that exposure to EoE inflammation limits tumor burden of esophageal carcinogenesis.DiscussionOur findings provide the first functional investigation of the relationship between EoE and esophageal cancer and suggest that esophageal epithelial remodeling events occurring in response to EoE inflammation may limit esophageal carcinogenesis. This investigation may have future implications for leveraging allergic inflammation-associated alterations in epithelial biology to prevent and/or treat esophageal cancer.

Barrett's esophagus (BE) is a common precancerous lesion that can progress to esophageal adenocarcinoma (EAC). There are significant alterations in the esophageal microbiome in the progression from healthy esophagus to BE to EAC, including an increased abundance of a variety of lactate-producing bacteria and an increase of lactate in the tumor microenvironment, as predicted by metabolic modeling. The role of bacterial lactate in EAC is unknown. Here, we utilize patient-derived organoid (PDO) models of EAC and demonstrate that lactate inhibits the growth and proliferation of EAC PDOs through alterations in the tumor NADH/NAD+ redox state. Further RNA sequencing of EAC PDOs identifies ID1 and RSAD2 as potential regulatory molecules crucial in mediating lactate's ability to suppress glycolysis and proliferation. Gene ontology analysis also identifies the activation of inflammatory and immunological pathways in addition to alterations in the metabolic pathways in EAC PDOs exposed to lactate, suggesting a multi-faceted role for lactate in the pathogenesis of EAC. [ABSTRACT FROM AUTHOR]

Background: Esophageal organoids from a variety of pathologies including cancer are grown in Advanced Dulbecco's Modified Eagle Medium-Nutrient Mixture F12 (hereafter ADF). However, the currently available ADF-based formulations are suboptimal for normal human esophageal organoids, limiting the ability to compare normal esophageal organoids with those representing a given disease state. Methods: We have utilized immortalized normal human esophageal epithelial cell (keratinocyte) lines EPC1 and EPC2 and endoscopic normal esophageal biopsies to generate three-dimensional (3D) organoids. To optimize the ADF-based medium, we evaluated the requirement of exogenous epidermal growth factor (EGF) and inhibition of transforming growth factor-(TGF)-β receptor-mediated signaling, both key regulators of the proliferation of human esophageal keratinocytes. We have modeled human esophageal epithelial pathology by stimulating esophageal 3D organoids with interleukin (IL)-13, an inflammatory cytokine, or UAB30, a novel pharmacological activator of retinoic acid signaling. Results: The formation of normal human esophageal 3D organoids was limited by excessive EGF and intrinsic TGFβ-receptor-mediated signaling. Optimized HOME0 improved normal human esophageal organoid formation. In the HOME0-grown organoids, IL-13 and UAB30 induced epithelial changes reminiscent of basal cell hyperplasia, a common histopathologic feature in broad esophageal disease conditions including eosinophilic esophagitis. Conclusions: HOME0 allows modeling of the homeostatic differentiation gradient and perturbation of the human esophageal epithelium while permitting a comparison of organoids from mice and other organs grown in ADF-based media. [ABSTRACT FROM AUTHOR]

Subepithelial fibrosis occurs in a subset of eosinophilic esophagitis (EoE) patients and is associated with esophageal stricture. While mechanisms driving EoE fibrosis remain incompletely understood, findings from experimental systems support roles for epithelial-fibroblast crosstalk in this type of tissue remodeling. The current paradigm presents EoE as a progressive fibrostenotic disease in which aged patients develop fibrosis as a function of disease chronicity. In the current study we provide evidence that altered epithelial biology in the aging esophagus may also contribute to EoE-associated fibrosis. We find that induction of EoE inflammation in young and aged mice using the MC903/Ovalbumin protocol for the same time period results in increased lamina propria thickness uniquely in aged animals. Additionally, epithelial cells from aged mice less efficiently limit fibroblast contractility in collagen plug contraction assays compared to those from their young counterparts. Finally, to identify potential mechanisms through which aged esophageal epithelial cells may stimulate fibrotic remodeling, we perform cytokine array experiments in young and aged mice. These studies are significant as identification of age-associated factors that contribute to fibrotic remodeling may aid in the design of strategies toward early detection, prevention, and therapy of fibrostenotic EoE.

Although activated Notch receptors have been associated with chemoresistance in cancer, the role of specific Notch ligands remain elusive. Here, the authors show that in breast cells the Notch ligand DLL1 is expressed in cells with a cancer stem cell phenotype and promote doxorubicin resistance in part through NF-kB, as well as metastasis.

Background & Aims: Oropharyngeal and esophageal squamous cell carcinomas, especially the latter, are a lethal disease, featuring intratumoral cancer cell heterogeneity and therapy resistance. To facilitate cancer therapy in personalized medicine, three-dimensional (3D) organoids may be useful for functional characterization of cancer cells ex vivo. We investigated the feasibility and the utility of patient-derived 3D organoids of esophageal and oropharyngeal squamous cell carcinomas. Methods: We generated 3D organoids from paired biopsies representing tumors and adjacent normal mucosa from therapy-naïve patients and cell lines. We evaluated growth and structures of 3D organoids treated with 5-fluorouracil ex vivo. Results: Tumor-derived 3D organoids were grown successfully from 15 out of 21 patients (71.4%) and passaged with recapitulation of the histopathology of the original tumors. Successful formation of tumor-derived 3D organoids was associated significantly with poor response to presurgical neoadjuvant chemotherapy or chemoradiation therapy in informative patients (P = 0.0357, progressive and stable diseases, n = 10 vs. partial response, n = 6). The 3D organoid formation capability and 5-fluorouracil resistance were accounted for by cancer cells with high CD44 expression and autophagy, respectively. Such cancer cells were found to be enriched in patient-derived 3D organoids surviving 5-fluorouracil treatment. Conclusions: The single cell-based 3D organoid system may serve as a highly efficient platform to explore cancer therapeutics and therapy resistance mechanisms in conjunction with morphological and functional assays with implications for translation in personalized medicine. Keywords: 3D Organoids, Autophagy, CD44, 5-Fluorouracil

Most colorectal cancers (CRCs) are moderately differentiated or well differentiated, a status that is preserved even in metastatic tumors. However, the molecular mechanisms underlying CRC differentiation remain to be elucidated. Herein, we unravel a potentially novel posttranscriptional regulatory mechanism via a LIN28B/CDX2 signaling axis that plays a critical role in mediating CRC differentiation. Owing to a large number of mRNA targets, the mRNA-binding protein LIN28B has diverse functions in development, metabolism, tissue regeneration, and tumorigenesis. Our RNA-binding protein IP (RIP) assay revealed that LIN28B directly binds CDX2 mRNA, which is a pivotal homeobox transcription factor in normal intestinal epithelial cell identity and differentiation. Furthermore, LIN28B overexpression resulted in enhanced CDX2 expression to promote differentiation in subcutaneous xenograft tumors generated from CRC cells and metastatic tumor colonization through mesenchymal-epithelial transition in CRC liver metastasis mouse models. A ChIP sequence for CDX2 identified α-methylacyl-CoA racemase (AMACR) as a potentially novel transcriptional target of CDX2 in the context of LIN28B overexpression. We also found that AMACR enhanced intestinal alkaline phosphatase activity, which is known as a key component of intestinal differentiation, through the upregulation of butyric acid. Overall, we demonstrated that LIN28B promotes CRC differentiation through the CDX2/AMACR axis.

During alcohol consumption, the esophageal mucosa is directly exposed to high concentrations of ethanol (EtOH). We therefore investigated the response of normal human esophageal epithelial cell lines EPC1, EPC2 and EPC3 to acute EtOH exposure. While these cells were able to tolerate 2% EtOH for 8 h in both three-dimensional organoids and monolayer culture conditions, RNA sequencing suggested that EtOH induced mitochondrial dysfunction. With EtOH treatment, EPC1 and EPC2 cells also demonstrated decreased mitochondrial ATPB protein expression by immunofluorescence and swollen mitochondria lacking intact cristae by transmission electron microscopy. Mitochondrial membrane potential (ΔΨm) was decreased in a subset of EPC1 and EPC2 cells stained with ΔΨm-sensitive dye MitoTracker Deep Red. In EPC2, EtOH decreased ATP level while impairing mitochondrial respiration and electron transportation chain functions, as determined by ATP fluorometric assay, respirometry, and liquid chromatography-mass spectrometry. Additionally, EPC2 cells demonstrated enhanced oxidative stress by flow cytometry for mitochondrial superoxide (MitoSOX), which was antagonized by the mitochondria-specific antioxidant MitoCP. Concurrently, EPC1 and EPC2 cells underwent autophagy following EtOH exposure, as evidenced by flow cytometry for Cyto-ID, which detects autophagic vesicles, and immunoblots demonstrating induction of the lipidated and cleaved form of LC3B and downregulation of SQSTM1/p62. In EPC1 and EPC2, pharmacological inhibition of autophagy flux by chloroquine increased mitochondrial oxidative stress while decreasing cell viability. In EPC2, autophagy induction was coupled with phosphorylation of AMP activated protein kinase (AMPK), a cellular energy sensor responding to low ATP levels, and dephosphorylation of downstream substrates of mechanistic Target of Rapamycin Complex (mTORC)-1 signaling. Pharmacological AMPK activation by AICAR decreased EtOH-induced reduction of ΔΨm and ATP in EPC2. Taken together, acute EtOH exposure leads to mitochondrial dysfunction and oxidative stress in esophageal keratinocytes, where the AMPK-mTORC1 axis may serve as a regulatory mechanism to activate autophagy to provide cytoprotection against EtOH-induced cell injury.

Aberrations in the esophageal proliferation-differentiation gradient are histologic hallmarks in eosinophilic esophagitis (EoE) and gastroesophageal reflux disease. A reliable protocol to grow 3-dimensional (3D) esophageal organoids is needed to study esophageal epithelial homeostasis under physiological and pathologic conditions. Methods: We modified keratinocyte-serum free medium to grow 3D organoids from endoscopic esophageal biopsies, immortalized human esophageal epithelial cells, and murine esophagi. Morphologic and functional characterization of 3D organoids was performed following genetic and pharmacologic modifications or exposure to EoE-relevant cytokines. The Notch pathway was evaluated by transfection assays and by gene expression analyses in vitro and in biopsies. Results: Both murine and human esophageal 3D organoids displayed an explicit proliferation-differentiation gradient. Notch inhibition accumulated undifferentiated basal keratinocytes with deregulated squamous cell differentiation in organoids. EoE patient-derived 3D organoids displayed normal epithelial structure ex vivo in the absence of the EoE inflammatory milieu. Stimulation of esophageal 3D organoids with EoE-relevant cytokines resulted in a phenocopy of Notch inhibition in organoid 3D structures with recapitulation of reactive epithelial changes in EoE biopsies, where Notch3 expression was significantly decreased in EoE compared with control subjects. Conclusions: Esophageal 3D organoids serve as a novel platform to investigate regulatory mechanisms in squamous epithelial homeostasis in the context of EoE and other diseases. Notch-mediated squamous cell differentiation is suppressed by cytokines known to be involved in EoE, suggesting that this may contribute to epithelial phenotypes associated with disease. Genetic and pharmacologic manipulations establish proof of concept for the utility of organoids for future studies and personalized medicine in EoE and other esophageal diseases.

The alcohol metabolite acetaldehyde is a potent human carcinogen linked to esophageal squamous cell carcinoma (ESCC) initiation and development. Aldehyde dehydrogenase 2 (ALDH2) is the primary enzyme that detoxifies acetaldehyde in the mitochondria. Acetaldehyde accumulation causes genotoxic stress in cells expressing the dysfunctional ALDH2E487K dominant negative mutant protein linked to ALDH2*2 , the single nucleotide polymorphism highly prevalent among East Asians. Heterozygous ALDH2*2 increases the risk for the development of ESCC and other alcohol-related cancers. Despite its prevalence and link to malignant transformation, how ALDH2 dysfunction influences ESCC pathobiology is incompletely understood. Herein, we characterize how ESCC and preneoplastic cells respond to alcohol exposure using cell lines, three-dimensional organoids and xenograft models. We find that alcohol exposure and ALDH2*2 cooperate to increase putative ESCC cancer stem cells with high CD44 expression (CD44H cells) linked to tumor initiation, repopulation and therapy resistance. Concurrently, ALHD2*2 augmented alcohol-induced reactive oxygen species and DNA damage to promote apoptosis in the non-CD44H cell population. Pharmacological activation of ALDH2 by Alda-1 inhibits this phenotype, suggesting that acetaldehyde is the primary driver of these changes. Additionally, we find that Aldh2 dysfunction affects the response to cisplatin, a chemotherapeutic commonly used for the treatment of ESCC. Aldh2 dysfunction facilitated enrichment of CD44H cells following cisplatin-induced oxidative stress and cell death in murine organoids, highlighting a potential mechanism driving cisplatin resistance. Together, these data provide evidence that ALDH2 dysfunction accelerates ESCC pathogenesis through enrichment of CD44H cells in response to genotoxic stressors such as environmental carcinogens and chemotherapeutic agents. [ABSTRACT FROM AUTHOR]

Background: Alcohol (ethanol) consumption is a major risk factor for head and neck and esophageal squamous cell carcinomas (SCCs). However, how ethanol (EtOH) affects SCC homeostasis is incompletely understood. Methods: We utilized three-dimensional (3D) organoids and xenograft tumor transplantation models to investigate how EtOH exposure influences intratumoral SCC cell populations including putative cancer stem cells defined by high CD44 expression (CD44H cells). Results: Using 3D organoids generated from SCC cell lines, patient-derived xenograft tumors, and patient biopsies, we found that EtOH is metabolized via alcohol dehydrogenases to induce oxidative stress associated with mitochondrial superoxide generation and mitochondrial depolarization, resulting in apoptosis of the majority of SCC cells within organoids. However, CD44H cells underwent autophagy to negate EtOH-induced mitochondrial dysfunction and apoptosis and were subsequently enriched in organoids and xenograft tumors when exposed to EtOH. Importantly, inhibition of autophagy increased EtOH-mediated apoptosis and reduced CD44H cell enrichment, xenograft tumor growth, and organoid formation rate. Conclusions: This study provides mechanistic insights into how EtOH may influence SCC cells and establishes autophagy as a potential therapeutic target for the treatment of EtOH-associated SCC.

Notch receptors can exert different roles in cancer. In this manuscript, the authors reveal that Notch1 activation and EMT promote tumor initiation and cancer cell heterogeneity in squamous cell carcinoma, while the repression of Notch3 by ZEB1 limits Notch1-induced differentiation, permitting Notch1-mediated EMT.