Autophagic state prospectively identifies facultative stem cells in the intestinal epithelium.

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.

Diclofenac exhibits cytotoxic activity associated with metabolic alterations and p53 induction in ESCC cell lines and decreases ESCC tumor burden in vivo.

Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive forms of human malignancy, often displaying limited therapeutic response. Here, we examine the non-steroidal anti-inflammatory drug diclofenac (DCF) as a novel therapeutic agent in ESCC using complementary in vitro and in vivo models. DCF selectively reduced viability of human ESCC cell lines TE11, KYSE150, and KYSE410 as compared with normal primary or immortalized esophageal keratinocytes. Apoptosis and altered cell cycle profiles were documented in DCF-treated TE11 and KYSE 150. In DCF-treated TE11, RNA-Sequencing identified differentially expressed genes and Ingenuity Pathway Analysis predicted alterations in pathways associated with cellular metabolism and p53 signaling. Downregulation of proteins associated with glycolysis was documented in DCF-treated TE11 and KYSE150. In response to DCF, TE11 cells further displayed reduced levels of ATP, pyruvate, and lactate. Evidence of mitochondrial depolarization and superoxide production was induced by DCF in TE11 and KYSE150. In DCF-treated TE11, the superoxide scavenger MitoTempo improved viability, supporting a role for mitochondrial reactive oxygen species in DCF-mediated toxicity. DCF treatment resulted in increased expression of p53 in TE11 and KYSE150. p53 was further identified as a mediator of DCF-mediated toxicity in TE11 as genetic depletion of p53 partially limited apoptosis in response to DCF. Consistent with the anticancer activity of DCF in vitro, the drug significantly decreased tumor burdene in syngeneic ESCC xenograft tumors and 4-nitroquinoline 1-oxide-mediated ESCC lesions in vivo. These preclinical findings identify DCF as an experimental therapeutic that should be explored further in ESCC.

Intestinal epithelial autophagy is required for the regenerative benefit of calorie restriction.

Calorie restriction can enhance the regenerative capacity of the injured intestinal epithelium. Among other metabolic changes, calorie restriction can activate the autophagy pathway. Although independent studies have attributed the regenerative benefit of calorie restriction to downregulation of mTORC1, it is not known whether autophagy itself is required for the regenerative benefit of calorie restriction. We used mouse and organoid models with autophagy gene deletion to evaluate the contribution of autophagy to intestinal epithelial regeneration following calorie restriction. In the absence of injury, mice with intestinal epithelial-specific deletion of autophagy gene Atg7 (Atg7ΔIEC) exhibit weight loss and histological changes similar to wild-type mice following calorie restriction. Conversely, calorie-restricted Atg7ΔIEC mice displayed a significant reduction in regenerative crypt foci after irradiation compared with calorie-restricted wild-type mice. Targeted analyses of tissue metabolites in calorie-restricted mice revealed an association between calorie restriction and reduced glycocholic acid (GCA) in wild-type mice but not in Atg7ΔIEC mice. To evaluate whether GCA can directly modulate epithelial stem cell self-renewal, we performed enteroid formation assays with or without GCA. Wild-type enteroids exhibited reduced enteroid formation efficiency in response to GCA treatment, suggesting that reduced availability of GCA during calorie restriction may be one mechanism by which calorie restriction favors epithelial regeneration in a manner dependent upon epithelial autophagy. Taken together, our data support the premise that intestinal epithelial Atg7 is required for the regenerative benefit of calorie restriction, due in part to its role in modulating luminal GCA with direct effects on epithelial stem cell self-renewal.NEW & NOTEWORTHY 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.

Roles for GADD45 in Development and Cancer.

The Growth Arrest and DNA Damage-inducible 45 (GADD45) family of proteins are critical stress sensors that mediate various cellular responses, including DNA repair, cell cycle arrest, and apoptosis. Here, we review current literature investigating GADD45 family members as they relate to normal development and carcinogenesis. We first describe how modulation of GADD45 in model organisms has facilitated our understanding of roles for GADD45 family members in development and homeostasis. We then review current literature exploring roles for GADD45 in human cancer, describing cancer-associated alterations in expression of GADD45 family members; tumor suppressive and tumor promoting functions attributed to GADD5; and roles for GADD45 in cancer therapy. In exploring roles for GADD45 in development, homeostasis, and carcinogenesis, we aim to provide an informational resource that both highlighst current knowledge on this topic while also noting key gaps in our understanding of the biology of GADD45 that may be filled in order to best guide the development of novel approaches to improve diagnosis, monitoring, and therapy of human malignancies.

Posttranscriptional regulation of colonic epithelial repair by RNA binding protein IMP1/IGF2BP1.

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 (Imp1ΔIEC ) was associated with better regeneration in mice after irradiation. Here, we report increased IMP1 expression in patients with Crohn's disease and ulcerative colitis. We demonstrate that Imp1ΔIEC mice exhibit enhanced recovery following dextran sodium sulfate (DSS)-mediated colonic injury. Imp1ΔIEC 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 Imp1ΔIEC 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.

Persistent Basal Cell Hyperplasia Is Associated With Clinical and Endoscopic Findings in Patients With Histologically Inactive Eosinophilic Esophagitis.

BACKGROUND & AIMS: Although eosinophil count is the standard used to monitor disease activity in patients with eosinophilic esophagitis (EoE), there are often disparities between patient-reported symptoms and eosinophil counts. We examined the prevalence of epithelial alterations, namely basal cell hyperplasia (BCH) and spongiosis, among patients with inactive EoE (eosinophil counts below 15 following therapy) and aimed to determine whether maintenance of these changes in epithelial morphology are associated with persistent clinical findings. METHODS: Esophageal biopsies of 243 patients (mean age, 16.9 years) undergoing routine endoscopy at the University of Pennsylvania were evaluated for epithelial BCH and spongiosis. Univariable analysis was used to calculate the association between epithelial changes and symptoms as well as endoscopic findings and peak eosinophil count. We validated our findings using data from a cohort of patients at the University of North Carolina. RESULTS: The discovery and validation cohorts each included patients with inactive EoE, based on histologic factors, but ongoing BCH and spongiosis. Ongoing BCH, but not spongiosis, in patients with inactive EoE was associated with symptoms (odds ratio, 2.14; 95% CI, 1.03-4.42; P = .041) and endoscopic findings (odds ratio, 7.10; 95% CI, 3.12-16.18; P < .001). CONCLUSIONS: In patients with EoE, the presence of BCH might indicate ongoing disease activity, independent of eosinophil count. This might account for the persistent symptoms in patients who are considered to be in remission based on histologic factors.

The potential for malignancy from atopic disorders and allergic inflammation: A systematic review and meta-analysis.

OBJECTIVE: While chronic inflammation is a well-established risk factor for malignancy, studies evaluating the relationship between allergic inflammation and cancer have revealed conflicting results. Here, we aimed to assess the association between allergic inflammation in the lung (asthma), skin (eczema) or oesophagus (eosinophilic oesophagitis; EoE) and cancer at the organ site. DESIGN: We conducted a systematic review of the literature to identify observational studies (case-control, cohort and cross-sectional) evaluating the association between asthma and lung cancer, eczema and skin cancer, or EoE and oesophageal cancer. Random-effects meta-analysis was performed to define pooled estimates of effects. DATA SOURCES: PubMed, EMBASE and Web of Science. ELIGIBILITY CRITERIA FOR SELECTION: Included studies evaluated the incidence of cancer. RESULTS: Thirty-two studies met the inclusion criteria, 27 in the lung, four in the skin and one in the oesophagus. Meta-analysis of the three studies with prospective data collection of asthma diagnosis revealed a positive association with incident lung cancer (OR 1.27, 95% CI 1.09-1.44); however, this result was not consistently supported by the larger dataset of retrospective studies (OR 1.37, 95% CI 0.90-1.83). Overall, studies in the lung displayed significant heterogeneity (I2 98%, P < .0001), but no significant effect modification on the association between asthma and lung cancer was identified for the variables of sex, smoking or study design. Meta-analysis could not be applied to the four papers reviewed in the skin, but three suggested an association between eczema and non-melanoma skin cancer, while the remaining study failed to identify an association between melanoma and eczema. A single study meeting inclusion criteria showed no association between EoE and oesophageal malignancy. CONCLUSIONS: The current data cannot exclude the possibility of an association between atopy and malignancy the lung, skin and oesophagus. The relationship between allergy and cancer should be explored further in prospective studies that any association identified between these conditions has the potential for significant public health implications.

Fibrostenotic eosinophilic esophagitis might reflect epithelial lysyl oxidase induction by fibroblast-derived TNF-α.

BACKGROUND: Fibrosis and stricture are major comorbidities in patients with eosinophilic esophagitis (EoE). Lysyl oxidase (LOX), a collagen cross-linking enzyme, has not been investigated in the context of EoE. OBJECTIVE: We investigated regulation of epithelial LOX expression as a novel biomarker and functional effector of fibrostenotic disease conditions associated with EoE. METHODS: LOX expression was analyzed by using RNA-sequencing, PCR assays, and immunostaining in patients with EoE; cytokine-stimulated esophageal 3-dimensional organoids; and fibroblast-epithelial cell coculture, the latter coupled with fluorescence-activated cell sorting. RESULTS: Gene ontology and pathway analyses linked TNF-α and LOX expression in patients with EoE, which was validated in independent sets of patients with fibrostenotic conditions. TNF-α-mediated epithelial LOX upregulation was recapitulated in 3-dimensional organoids and coculture experiments. We find that fibroblast-derived TNF-α stimulates epithelial LOX expression through activation of nuclear factor κB and TGF-ß-mediated signaling. In patients receiver operating characteristic analyses suggested that LOX upregulation indicates disease complications and fibrostenotic conditions in patients with EoE. CONCLUSIONS: There is a novel positive feedback mechanism in epithelial LOX induction through fibroblast-derived TNF-α secretion. Esophageal epithelial LOX might have a role in the development of fibrosis with substantial translational implications.

RNA regulons are essential in intestinal homeostasis.

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.

Autophagy mediates epithelial cytoprotection in eosinophilic oesophagitis.

OBJECTIVE: The influence of eosinophilic oesophagitis (EoE)-associated inflammation upon oesophageal epithelial biology remains poorly understood. We investigated the functional role of autophagy in oesophageal epithelial cells (keratinocytes) exposed to the inflammatory EoE milieu. DESIGN: Functional consequences of genetic or pharmacological autophagy inhibition were assessed in endoscopic oesophageal biopsies, human oesophageal keratinocytes, single cell-derived ex vivo murine oesophageal organoids as well as a murine model recapitulating EoE-like inflammation and basal cell hyperplasia. Gene expression, morphological and functional characterisation of autophagy and oxidative stress were performed by transmission electron microscopy, immunostaining, immunoblotting, live cell imaging and flow cytometry. RESULTS: EoE-relevant inflammatory conditions promoted autophagy and basal cell hyperplasia in three independent murine EoE models and oesophageal organoids. Inhibition of autophagic flux via chloroquine treatment augmented basal cell hyperplasia in these model systems. Oesophageal keratinocytes stimulated with EoE-relevant cytokines, including tumour necrosis factor-α and interleukin-13 exhibited activation of autophagic flux in a reactive oxygen species-dependent manner. Autophagy inhibition via chloroquine treatment or depletion of Beclin-1 or ATG-7, augmented oxidative stress induced by EoE-relevant stimuli in murine EoE, oesophageal organoids and human oesophageal keratinocytes. Oesophageal epithelia of paediatric EoE patients with active inflammation displayed increased autophagic vesicle content compared with normal and EoE remission subjects. Functional flow cytometric analysis revealed autophagic flux in human oesophageal biopsies. CONCLUSIONS: Our findings reveal for the first time that autophagy may function as a cytoprotective mechanism to maintain epithelial redox balance and homeostasis under EoE inflammation-associated stress, providing mechanistic insights into the role of autophagy in EoE pathogenesis.

Fibroblast growth factor-2-mediated FGFR/Erk signaling supports maintenance of cancer stem-like cells in esophageal squamous cell carcinoma.

In esophageal squamous cell carcinoma (ESCC), a subset of cells defined by high expression of CD44 and low expression of CD24 has been reported to possess characteristics of cancer stem-like cells (CSCs). Novel therapies directly targeting CSCs have the potential to improve prognosis of ESCC patients. Although fibroblast growth factor-2 (FGF-2) expression correlates with recurrence and poor survival in ESCC patients, the role of FGF-2 in regulation of ESCC CSCs has yet to be elucidated. We report that FGF-2 is significantly upregulated in CSCs and significantly increases CSC content in ESCC cell lines by inducing epithelial-mesenchymal transition (EMT). Conversely, the FGFR inhibitor, AZD4547, sharply diminishes CSCs via induction of mesenchymal-epithelial transition. Further experiments revealed that MAPK/Erk kinase (Mek)/extracellular signal-regulated kinases (Erk) pathway is crucial for FGF-2-mediated CSC regulation. Pharmacological inhibition of FGF receptor (FGFR)-mediated signaling via AZD4547 did not affect CSCs in Ras mutated cells, implying that Mek/Erk pathway, downstream of FGFR signaling, might be an important regulator of CSCs. Indeed, the Mek inhibitor, trametinib, efficiently suppressed ESCC CSCs even in the context of Ras mutation. Consistent with these findings in vitro, xenotransplantation studies demonstrated that inhibition of FGF-2-mediated FGFR/Erk signaling significantly delayed tumor growth. Taken together, these findings indicate that FGF-2 is an essential factor regulating CSCs via Mek/Erk signaling in ESCC. Additionally, inhibition of FGFR and/or Mek signaling represents a potential novel therapeutic option for targeting CSCs in ESCC.

Autophagy levels are elevated in barrett's esophagus and promote cell survival from acid and oxidative stress.

Autophagy is a highly conserved mechanism that is activated during cellular stress. We hypothesized that autophagy may be induced by acid reflux, which causes injury, and inflammation, and therefore, contributes to the pathogenesis of Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC). Currently, the role of autophagy in BE and EAC is poorly studied. We quantitatively define autophagy levels in human BE cell lines, a transgenic mouse model of BE, and human BE, and EAC biopsies. Human non-dysplastic BE had the highest basal number of autophagic vesicles (AVs), while AVs were reduced in normal squamous cells and dysplastic BE cells, and nearly absent in EAC. To demonstrate a functional role for autophagy in BE pathogenesis, normal squamous (STR), non-dysplastic BE (CPA), dysplastic BE (CPD), and EAC (OE19) cell lines were exposed to an acid pulse (pH 3.5) followed by incubation in the presence or absence of chloroquine, an autophagy inhibitor. Acid exposure increased reactive oxygen species (ROS) levels in STR and CPA cells. Chloroquine alone had a small impact on intracellular ROS or cell survival. However, combination of chloroquine with the acid pulse resulted in a significant increase in ROS levels at 6 h in STR and CPA cells, and increased cell death in all cell lines. These findings establish increased numbers of AVs in human BE compared to normal squamous or EAC, and suggest that autophagy functions to improve cell survival after acid reflux injury. Autophagy may thus play a critical role in BE pathogenesis and progression. © 2015 Wiley Periodicals, Inc.

Eosinophilic Esophagitis-Associated Chemical and Mechanical Microenvironment Shapes Esophageal Fibroblast Behavior.

OBJECTIVES: Eosinophilic esophagitis (EoE) is an immune-mediated allergic disease characterized by progressive esophageal dysmotility and fibrotic stricture associated with chronic esophageal fibroblast activation. It remains unknown how esophageal fibroblasts respond to EoE-relevant matrix stiffness or inflammatory cytokines. METHODS: Immunofluorescence was used to evaluate α-smooth muscle actin (α-SMA) expression in endoscopic esophageal biopsies. Primary esophageal fibroblasts from adult and pediatric patients with or without EoE were exposed to transforming growth factor (TGF)ß to determine gene expression, collagen-matrix contractility, and cytoskeletal organization. The influence of matrix stiffness upon fibroblast behavior was assessed on the engineered surface of polyacrylamide gels with varying stiffness. Fibroblast traction forces were measured using microfabricated-post-array-detectors. RESULTS: EoE esophageal fibroblasts had enhanced α-SMA expression. TGFß not only stimulated enhanced fibroblast-specific gene expression but also promoted fibroblast-mediated collagen-matrix contraction, despite disease state or age of patients as the origin of cells. Unlike conventional monolayer cell, culture conditions using plastic surface (1 GPa) that activates fibroblasts constitutively, our engineered platforms recapitulating physiologically relevant stiffness (1-20 kPa) revealed that matrix stiffness defines the extent of α-SMA expression, intracellular collagen fibril organization, SMAD3 phosphorylation, and fibroblast traction force. CONCLUSIONS: Matrix stiffness may critically influence TGFß-mediated gene expression and functions of esophageal fibroblasts ex vivo independent of age and disease conditions. These findings provide a novel insight into the pathogenesis of fibrostenotic disease in EoE.

WNT10A promotes an invasive and self-renewing phenotype in esophageal squamous cell carcinoma.

Esophageal cells overexpressing epidermal growth factor receptor (EGFR) and TP53 mutation can invade into the extracellular matrix when grown in 3D-organotypic cultures (OTC) and mimic early invasion in esophageal squamous cell carcinoma (ESCC). We have performed laser capture microdissection with RNA microarray analysis on the invasive and non-invasive tumor cells of p53(R175H)-overexpressing OTC samples to determine candidate genes facilitating tumor invasion. WNT10A was found to be >4-fold upregulated in the invasive front. Since WNT10A is also prominently upregulated during placode promotion in hair follicle development, a process that requires epithelial cells to thicken and elongate, in order to allow downward growth, we hypothesized that WNT10A may be important in mediating a similar mechanism of tumor cell invasion in ESCC. We have found that WNT10A expression is significantly upregulated in human ESCC, when compared with normal adjacent tissue. Furthermore, high WNT10A expression levels correlate with poor survival. Interestingly, we observe that WNT10A is expressed early in embryogenesis, but is reduced dramatically postnatally. We demonstrate that overexpression of WNT10a promotes migration and invasion, and proliferation of transformed esophageal cells. Lastly, we show that WNT10A overexpression induces a greater CD44(High)/CD24(Low) population, which are putative markers of cancer stem cells, and increases self-renewal capability. Taken together, we propose that WNT10A acts as an oncofetal factor that is highly expressed and may promote proper development of the esophagus. During tumorigenesis, it is aberrantly overexpressed in order to promote ESCC migration and invasion, and may be linked to self-renewal of a subset of ESCC cells.

The oncogene HER2/neu (ERBB2) requires the hypoxia-inducible factor HIF-1 for mammary tumor growth and anoikis resistance.

ERBB2, a receptor tyrosine kinase amplified in breast cancer, is a well established regulator of tumor growth in vivo and anoikis resistance leading to disruption of architecture in three-dimensional mammary epithelial acinar structures in vitro. ERBB2 promotes anoikis resistance by maintaining signaling pathways and by rescuing metabolic defects and thus inhibiting accumulation of deleterious reactive oxygen species. Recent evidence suggests that hypoxia, via hypoxia-inducible factors (HIFs), can inhibit anoikis; thus, we hypothesized that HIF-1 may play a role in ERBB2-mediated anoikis resistance and oncogenesis. Indeed, tumors isolated from MMTV-Neu mice contain elevated HIF-1α levels and tumor cells created from MMTV-Neu mice harboring deletion of Hif1α alleles reduced primary tumor growth in vivo. ERBB2 overexpressing cancer cells stabilize HIF under normoxic conditions and require HIF-1 for ERBB2-mediated anchorage-independence, three-dimensional culture growth and anoikis resistance. HIF-1 reduction in ERBB2 cells was associated with induction of the pro-anoikis protein BIM and decreased ERK and AKT signaling during cell detachment. ERBB2-mediated inhibition of metabolic defects, including decreased reactive oxygen species generation in suspension, required HIF-1 expression that was critical for ERBB2-mediated oncogenesis. Gene expression profiling of hypoxic three-dimensional acinar structures identified a number of genes elevated in response to hypoxia that are known ERBB2 targets, suggesting that hypoxic conditions and ERBB2 overexpression share both phenotypic and genetic components via HIF-1 regulation. Thus, our data demonstrate that ERBB2 requires HIF-1 for tumor growth and suggest that HIF is a major downstream regulator of ERBB2 that protects cells from anoikis and metabolic stress caused by decreased matrix adhesion.

Alginate encapsulation supports the growth and differentiation of human primordial follicles within ovarian cortical tissue.

PURPOSE: In vitro follicle growth (IVFG) is an investigational fertility preservation technique in which immature follicles are grown in culture to produce mature eggs that can ultimately be fertilized. Although progress has been made in growing primate primary and secondary follicles in vitro, it has been a relatively greater challenge to isolate and culture primordial follicles. The purpose of this study was to develop methods to grow human primordial follicles in vitro using alginate hydrogels. METHODS: We obtained human ovarian tissue for research purposes through the National Physicians Cooperative from nationwide sites and used it to test two methods for culturing primordial follicles. First, primordial follicles were isolated from the ovarian cortex and encapsulated in alginate hydrogels. Second, 1 mm × 1 mm pieces of 500 µm-thick human ovarian cortex containing primordial follicles were encapsulated in alginate hydrogels, and survival and follicle development within the tissue was assessed for up to 6 weeks. RESULTS: We found that human ovarian tissue could be kept at 4 °C for up to 24 h while still maintaining follicle viability. Primordial follicles isolated from ovarian tissue did not survive culture. However, encapsulation and culture of ovarian cortical pieces supported the survival, differentiation, and growth of primordial and primary follicles. Within several weeks of culture, many of the ovarian tissue pieces had formed a defined surface epithelium and contained growing preantral and antral follicles. CONCLUSIONS: The early stages of in vitro human follicle development require the support of the native ovarian cortex.

Epithelial-Fibroblast Crosstalk in Eosinophilic Esophagitis.

Eosinophilic esophagitis (EoE) is an emerging form of food allergy that exerts a significant clinical and financial burden worldwide. EoE is clinically characterized by eosinophil-rich inflammatory infiltrates in esophageal mucosa and esophageal dysfunction. Remodeling events in esophageal epithelium and lamina propria also frequently occur in patients with EoE. Because subepithelial fibrosis is associated with esophageal stricture, the most severe consequence of EoE, there exists an urgent need for a deeper understanding of the molecular mechanisms mediating fibrosis in EoE. Here, we review emerging evidence from experimental model systems that implicates crosstalk between esophageal epithelial cells and underlying stromal cells in EoE fibrosis. We further discuss implications for epithelial-stromal interaction with regard to EoE patient care and propose future directions that may be pursued to further the understanding of epithelial-stromal crosstalk in EoE pathobiology.

Esophageal epithelial Ikkß deletion promotes eosinophilic esophagitis in experimental allergy mouse model.

Background: Eosinophilic esophagitis (EoE) is a chronic T helper type 2 (Th2)-associated inflammatory disorder triggered by food allergens, resulting in esophageal dysfunction through edema, fibrosis, and tissue remodeling. The role of epithelial remodeling in EoE pathogenesis is critical but not fully understood. Objective: To investigate the role of epithelial IKKß/NFκB signaling in EoE pathogenesis using a mouse model with conditional Ikk ß knockout in esophageal epithelial cells ( Ikk ß EEC-KO ). Methods: EoE was induced in Ikkß EEC-KO mice through skin sensitization with MC903/Ovalbumin (OVA) followed by intraesophageal OVA challenge. Histological and transcriptional analyses were performed to assess EoE features. Single-cell RNA sequencing (scRNA-seq) was used to profile esophageal mucosal cell populations and gene expression changes. Results: Ikkß EEC-KO /EoE mice exhibited hallmark EoE features, including eosinophil infiltration, intraepithelial eosinophils, microabscesses, basal cell hyperplasia, and lamina propria remodeling. RNA-seq revealed significant alterations in IKKß/NFκB signaling pathways, with decreased expression of RELA and increased expression of IKKß negative regulators. scRNA- seq analyses identified disrupted epithelial differentiation and barrier integrity, alongside increased type 2 immune responses and peptidase activity. Conclusion: Our study demonstrates that loss of epithelial IKKß signaling exacerbates EoE pathogenesis, highlighting the critical role of this pathway in maintaining epithelial homeostasis and preventing allergic inflammation. The Ikkß EEC-KO /EoE mouse model closely mirrors human EoE, providing a valuable tool for investigating disease mechanisms and therapeutic targets. This model can facilitate the development of strategies to prevent chronic inflammation and tissue remodeling in EoE. Key Messages: Critical Role of Epithelial IKKß/NFκB Signaling: Loss of this signaling exacerbates EoE, causing eosinophil infiltration, basal cell hyperplasia, and tissue remodeling, highlighting its importance in esophageal health.Molecular Insights and Therapeutic Targets: scRNA-seq identified disrupted epithelial differentiation, barrier integrity, and enhanced type 2 immune responses, suggesting potential therapeutic targets for EoE. Relevance of the Ikkß EEC-KO /EoE Mouse Model: This model replicates human EoE features, making it a valuable tool for studying EoE mechanisms and testing treatments, which can drive the development of effective therapies. Capsule Summary: This study reveals the crucial role of epithelial IKKß/NFκB signaling in EoE, providing insights into disease mechanisms and potential therapeutic targets, highly relevant for advancing clinical management of EoE.

Derivation of human primary prostate epithelial cell lines by differentially targeting the CDKN2A locus along with expression of hTERT.

Prostate cancer (PCa) is the most common cancer diagnosed in men worldwide and the second leading cause of cancer-related deaths in US males in 2022. Prostate cancer also represents the second highest cancer mortality disparity between non-Hispanic blacks and whites. However, there is a relatively small number of prostate normal and cancer cell lines compared to other cancers. To identify the molecular basis of PCa progression, it is important to have prostate epithelial cell (PrEC) lines as karyotypically normal as possible. Our lab recently developed a novel methodology for the rapid and efficient immortalization of normal human PrEC that combines simultaneous CRISPR-directed inactivation of CDKN2A exon 2 (which directs expression of p16INK4A and p14ARF) and ectopic expression of an hTERT transgene. To optimize this methodology to generate immortalized lines with minimal genetic alterations, we sought to target exon 1α of the CDKN2A locus so that p16INK4A expression is ablated while p14ARF expression remains unaltered. Here we describe the establishment of two cell lines: one with the above-mentioned p16INK4A only loss, and a second line targeting both products in the CDKN2A locus. We characterize the potential lineage origin of these new cell lines along with our previously obtained clones, revealing distinct gene expression signatures. Based on the analyses of protein markers and RNA expression signatures, these cell lines are most closely related to a subpopulation of basal prostatic cells. Given the simplicity of this one-step methodology and the fact that it uses only the minimal genetic alterations necessary for immortalization, it should also be suitable for the establishment of cell lines from primary prostate tumor samples, an urgent need given the limited number of available prostate cancer cell lines.