Publications by authors named "Jianwen Que"

74 Publications

Helicobacter pylori pathogen inhibits cellular responses to oncogenic stress and apoptosis.

PLoS Pathog 2022 06 29;18(6):e1010628. Epub 2022 Jun 29.

Department of Surgery, Miller School of Medicine, University of Miami, Miami, Florida, United States of America.

Helicobacter pylori (H. pylori) is a common gastric pathogen that infects approximately half of the world's population. Infection with H. pylori can lead to diverse pathological conditions, including chronic gastritis, peptic ulcer disease, and cancer. The latter is the most severe consequence of H. pylori infection. According to epidemiological studies, gastric infection with H. pylori is the strongest known risk factor for non-cardia gastric cancer (GC), which remains one of the leading causes of cancer-related deaths worldwide. However, it still remains to be poorly understood how host-microbe interactions result in cancer development in the human stomach. Here we focus on the H. pylori bacterial factors that affect the host ubiquitin proteasome system. We investigated E3 ubiquitin ligases SIVA1 and ULF that regulate p14ARF (p19ARF in mice) tumor suppressor. ARF plays a key role in regulation of the oncogenic stress response and is frequently inhibited during GC progression. Expression of ARF, SIVA1 and ULF proteins were investigated in gastroids, H. pylori-infected mice and human gastric tissues. The role of the H. pylori type IV secretion system was assessed using various H. pylori isogenic mutants. Our studies demonstrated that H. pylori infection results in induction of ULF, decrease in SIVA1 protein levels, and subsequent ubiquitination and degradation of p14ARF tumor suppressor. Bacterial CagA protein was found to sequentially bind to SIVA1 and ULF proteins. This process is regulated by CagA protein phosphorylation at the EPIYA motifs. Downregulation of ARF protein leads to inhibition of cellular apoptosis and oncogenic stress response that may promote gastric carcinogenesis.
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http://dx.doi.org/10.1371/journal.ppat.1010628DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9242521PMC
June 2022

Activation of NOTCH signaling via DLL1 is mediated by APE1-redox-dependent NF-κB activation in oesophageal adenocarcinoma.

Gut 2022 Jun 24. Epub 2022 Jun 24.

Department of Surgery, University of Miami Miller School of Medicine, Miami, Florida, USA

Objective: Oesophageal adenocarcinoma (EAC) arises in the setting of Barrett's oesophagus, an intestinal metaplastic precursor lesion that can develop in patients with chronic GERD. Here, we investigated the role of acidic bile salts, the mimicry of reflux, in activation of NOTCH signaling in EAC.

Design: This study used public databases, EAC cell line models, L2-IL1β transgenic mouse model and human EAC tissue samples to identify mechanisms of NOTCH activation under reflux conditions.

Results: Analysis of public databases demonstrated significant upregulation of NOTCH signaling components in EAC. In vitro studies demonstrated nuclear accumulation of active NOTCH1 cleaved fragment (NOTCH intracellular domain) and upregulation of NOTCH targets in EAC cells in response to reflux conditions. Additional investigations identified DLL1 as the predominant ligand contributing to NOTCH1 activation under reflux conditions. We discovered a novel crosstalk between APE1 redox function, reflux-induced inflammation and DLL1 upregulation where NF-κB can directly bind to and induce the expression of DLL1. The APE1 redox function was crucial for activation of the APE1-NF-κB-NOTCH axis and promoting cancer cell stem-like properties in response to reflux conditions. Overexpression of APE1 and DLL1 was detected in gastro-oesophageal junctions of the L2-IL1ß transgenic mouse model and human EAC tissue microarrays. DLL1 high levels were associated with poor overall survival in patients with EAC.

Conclusion: These findings underscore a unique mechanism that links redox balance, inflammation and embryonic development (NOTCH) into a common pro-tumorigenic pathway that is intrinsic to EAC cells.
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http://dx.doi.org/10.1136/gutjnl-2022-327076DOI Listing
June 2022

Targeting the SOX2/PARP1 complex to intervene in the growth of esophageal squamous cell carcinoma.

Biomed Pharmacother 2022 Jun 20;153:113309. Epub 2022 Jun 20.

Central Laboratory, Xiang'an Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, Fujian 361102, China; School of Medicine, Xiamen University, Xiamen, Fujian 361102, China. Electronic address:

Elevated SOX2 protein levels are closely correlated with the increased incidence of esophageal squamous cell carcinoma (ESCC). However, establishing effective target measures for ESCC treatments continue to be researched. It has been previously proposed that SOX2 represents a potential therapeutic target for ESCC. Here, we found that the enzyme Poly(ADP-Ribose) polymerase 1 (PARP1) enriched in ESCCs interact with SOX2. Inhibition of PARP1 with 3-aminobenzamide (3-ABA) or shRNA knockdown reduced the proliferation of ESCCs, accompanied by decreased protein levels of SOX2. RNA sequencing demonstrated that PARP1 inhibition affected multiple signaling pathways involved in cancer cell proliferation. Additionally, 3-ABA synergistically suppressed the growth of ESCC cells when combined with cisplatin, and metformin potentiated the suppressive effect of 3-ABA on ESCC cell growth. Together these findings suggest that targeting SOX2 binding partner PARP1 provides a possible avenue to treat patients with high levels of SOX2.
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http://dx.doi.org/10.1016/j.biopha.2022.113309DOI Listing
June 2022

Caspase-4/11 exacerbates disease severity in SARS-CoV-2 infection by promoting inflammation and immunothrombosis.

Proc Natl Acad Sci U S A 2022 05 19;119(21):e2202012119. Epub 2022 May 19.

Department of Microbiology and Immunology, Emory University, Atlanta, GA 30322.

Severe acute respiratory syndrome coronavirus 2 (SARS–CoV-2) is a worldwide health concern, and new treatment strategies are needed. Targeting inflammatory innate immunity pathways holds therapeutic promise, but effective molecular targets remain elusive. Here, we show that human caspase-4 (CASP4) and its mouse homolog, caspase-11 (CASP11), are up-regulated in SARS–CoV-2 infections and that CASP4 expression correlates with severity of SARS–CoV-2 infection in humans. SARS–CoV-2–infected Casp11−/− mice were protected from severe weight loss and lung pathology, including blood vessel damage, compared to wild-type (WT) mice and mice lacking the caspase downstream effector gasdermin-D (Gsdmd−/−). Notably, viral titers were similar regardless of CASP11 knockout. Global transcriptomics of SARS–CoV-2–infected WT, Casp11−/−, and Gsdmd−/− lungs identified restrained expression of inflammatory molecules and altered neutrophil gene signatures in Casp11−/− mice. We confirmed that protein levels of inflammatory mediators interleukin (IL)-1β, IL-6, and CXCL1, as well as neutrophil functions, were reduced in Casp11−/− lungs. Additionally, Casp11−/− lungs accumulated less von Willebrand factor, a marker for endothelial damage, but expressed more Kruppel-Like Factor 2, a transcription factor that maintains vascular integrity. Overall, our results demonstrate that CASP4/11 promotes detrimental SARS–CoV-2–induced inflammation and coagulopathy, largely independently of GSDMD, identifying CASP4/11 as a promising drug target for treatment and prevention of severe COVID-19.
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http://dx.doi.org/10.1073/pnas.2202012119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9173818PMC
May 2022

Epithelial Wntless regulates postnatal alveologenesis.

Development 2022 01 10;149(1). Epub 2022 Jan 10.

Division of Digestive and Liver Diseases, Department of Medicine, Columbia Center for Human Development, Columbia University Medical Center, New York, NY 10032, USA.

Alveologenesis requires the coordinated modulation of the epithelial and mesenchymal compartments to generate mature alveolar saccules for efficient gas exchange. However, the molecular mechanisms underlying the epithelial-mesenchymal interaction during alveologenesis are poorly understood. Here, we report that Wnts produced by epithelial cells are crucial for neonatal alveologenesis. Deletion of the Wnt chaperone protein Wntless homolog (Wls) disrupts alveolar formation, resulting in enlarged saccules in Sftpc-Cre/Nkx2.1-Cre; Wlsloxp/loxp mutants. Although commitment of the alveolar epithelium is unaffected, α-SMA+ mesenchymal cells persist in the alveoli, accompanied by increased collagen deposition, and mutants exhibit exacerbated fibrosis following bleomycin challenge. Notably, α-SMA+ cells include a significant number of endothelial cells resembling endothelial to mesenchymal transition (EndMT), which is also present in Ager-CreER; Wlsloxp/loxp mutants following early postnatal Wls deletion. These findings provide initial evidence that epithelial-derived Wnts are crucial for the differentiation of the surrounding mesenchyme during early postnatal alveologenesis.
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http://dx.doi.org/10.1242/dev.199505DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8881739PMC
January 2022

Disruption of respiratory epithelial basement membrane in COVID-19 patients.

Mol Biomed 2021 20;2(1). Epub 2021 Mar 20.

Department of Medicine, Division of Pulmonary and Critical Care Medicine, Women's Guild Lung Institute, Cedars-Sinai Medical Center, Los Angeles, California USA.

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http://dx.doi.org/10.1186/s43556-021-00031-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7979449PMC
March 2021

Stem cells and origins of cancer in the upper gastrointestinal tract.

Cell Stem Cell 2021 08 14;28(8):1343-1361. Epub 2021 Jun 14.

Division of Digestive and Liver Diseases, Department of Medicine, Columbia University, College of Physicians and Surgeons, 1130 St. Nicholas Avenue, New York, NY 10032, USA; Herbert Irving Comprehensive Cancer Center, 1130 St. Nicholas Avenue, New York, NY 10032, USA. Electronic address:

The esophagus and stomach, joined by a unique transitional zone, contain actively dividing epithelial stem cells required for organ homeostasis. Upon prolonged inflammation, epithelial cells in both organs can undergo a cell fate switch leading to intestinal metaplasia, predisposing to malignancy. Here we discuss the biology of gastroesophageal stem cells and their role as cells of origin in cancer. We summarize the interactions between the stromal niche and gastroesophageal stem cells in metaplasia and early expansion of mutated stem-cell-derived clones during carcinogenesis. Finally, we review new approaches under development to better study gastroesophageal stem cells and advance the field.
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http://dx.doi.org/10.1016/j.stem.2021.05.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8844710PMC
August 2021

SARS-CoV-2 Nsp14 activates NF-κB signaling and induces IL-8 upregulation.

bioRxiv 2021 May 26. Epub 2021 May 26.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection leads to NF-κB activation and induction of pro-inflammatory cytokines, though the underlying mechanism for this activation is not fully understood. Our results reveal that the SARS-CoV-2 Nsp14 protein contributes to the viral activation of NF-κB signaling. Nsp14 caused the nuclear translocation of NF-κB p65. Nsp14 induced the upregulation of IL-6 and IL-8, which also occurred in SARS-CoV-2 infected cells. IL-8 upregulation was further confirmed in lung tissue samples from COVID-19 patients. A previous proteomic screen identified the putative interaction of Nsp14 with host Inosine-5'-monophosphate dehydrogenase 2 (IMPDH2) protein, which is known to regulate NF-κB signaling. We confirmed the Nsp14-IMPDH2 protein interaction and found that IMPDH2 knockdown or chemical inhibition using ribavirin (RIB) and mycophenolic acid (MPA) abolishes Nsp14-mediated NF-κB activation and cytokine induction. Furthermore, IMDPH2 inhibitors (RIB, MPA) efficiently blocked SARS-CoV-2 infection, indicating that IMDPH2, and possibly NF-κB signaling, is beneficial to viral replication. Overall, our results identify a novel role of SARS-CoV-2 Nsp14 in causing the activation of NF-κB.
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http://dx.doi.org/10.1101/2021.05.26.445787DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8168382PMC
May 2021

VEGF receptor 2 (KDR) protects airways from mucus metaplasia through a Sox9-dependent pathway.

Dev Cell 2021 06 18;56(11):1646-1660.e5. Epub 2021 May 18.

Columbia Center for Human Development & Division of Digestive and Liver Disease, Department of Medicine, Columbia University Medical Center, NY 10032, USA; Herbert Irving Comprehensive Cancer Center, Columbia University Medical Center, NY 10032, USA. Electronic address:

Mucus-secreting goblet cells are the dominant cell type in pulmonary diseases, e.g., asthma and cystic fibrosis (CF), leading to pathologic mucus metaplasia and airway obstruction. Cytokines including IL-13 are the major players in the transdifferentiation of club cells into goblet cells. Unexpectedly, we have uncovered a previously undescribed pathway promoting mucous metaplasia that involves VEGFa and its receptor KDR. Single-cell RNA sequencing analysis coupled with genetic mouse modeling demonstrates that loss of epithelial VEGFa, KDR, or MEK/ERK kinase promotes excessive club-to-goblet transdifferentiation during development and regeneration. Sox9 is required for goblet cell differentiation following Kdr inhibition in both mouse and human club cells. Significantly, airway mucous metaplasia in asthmatic and CF patients is also associated with reduced KDR signaling and increased SOX9 expression. Together, these findings reveal an unexpected role for VEGFa/KDR signaling in the defense against mucous metaplasia, offering a potential therapeutic target for this common airway pathology.
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http://dx.doi.org/10.1016/j.devcel.2021.04.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8187331PMC
June 2021

Role of Bacterial and Viral Pathogens in Gastric Carcinogenesis.

Cancers (Basel) 2021 Apr 14;13(8). Epub 2021 Apr 14.

Department of Surgery, University of Miami, Miami, FL 33136, USA.

Gastric cancer (GC) is one of the deadliest malignancies worldwide. In contrast to many other tumor types, gastric carcinogenesis is tightly linked to infectious events. Infections with () bacterium and Epstein-Barr virus (EBV) are the two most investigated risk factors for GC. These pathogens infect more than half of the world's population. Fortunately, only a small fraction of infected individuals develops GC, suggesting high complexity of tumorigenic processes in the human stomach. Recent studies suggest that the multifaceted interplay between microbial, environmental, and host genetic factors underlies gastric tumorigenesis. Many aspects of these interactions still remain unclear. In this review, we update on recent discoveries, focusing on the roles of various gastric pathogens and gastric microbiome in tumorigenesis.
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http://dx.doi.org/10.3390/cancers13081878DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8070847PMC
April 2021

A molecular single-cell lung atlas of lethal COVID-19.

Nature 2021 07 29;595(7865):114-119. Epub 2021 Apr 29.

Human Immune Monitoring Core, Columbia University Irving Medical Center, New York, NY, USA.

Respiratory failure is the leading cause of death in patients with severe SARS-CoV-2 infection, but the host response at the lung tissue level is poorly understood. Here we performed single-nucleus RNA sequencing of about 116,000 nuclei from the lungs of nineteen individuals who died of COVID-19 and underwent rapid autopsy and seven control individuals. Integrated analyses identified substantial alterations in cellular composition, transcriptional cell states, and cell-to-cell interactions, thereby providing insight into the biology of lethal COVID-19. The lungs from individuals with COVID-19 were highly inflamed, with dense infiltration of aberrantly activated monocyte-derived macrophages and alveolar macrophages, but had impaired T cell responses. Monocyte/macrophage-derived interleukin-1β and epithelial cell-derived interleukin-6 were unique features of SARS-CoV-2 infection compared to other viral and bacterial causes of pneumonia. Alveolar type 2 cells adopted an inflammation-associated transient progenitor cell state and failed to undergo full transition into alveolar type 1 cells, resulting in impaired lung regeneration. Furthermore, we identified expansion of recently described CTHRC1 pathological fibroblasts contributing to rapidly ensuing pulmonary fibrosis in COVID-19. Inference of protein activity and ligand-receptor interactions identified putative drug targets to disrupt deleterious circuits. This atlas enables the dissection of lethal COVID-19, may inform our understanding of long-term complications of COVID-19 survivors, and provides an important resource for therapeutic development.
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http://dx.doi.org/10.1038/s41586-021-03569-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8814825PMC
July 2021

Activation of NRF2 by APE1/REF1 is redox-dependent in Barrett's related esophageal adenocarcinoma cells.

Redox Biol 2021 07 19;43:101970. Epub 2021 Apr 19.

Department of Surgery, Miller School of Medicine, University of Miami, Miami, FL, 33136, USA; Sylvester Comprehensive Cancer Center, Miller School of Medicine, University of Miami, Miami, FL-33136, USA; Department of Veterans Affairs, Miami Healthcare System, Miami, FL, 33136, USA. Electronic address:

Background: Chronic gastroesophageal reflux disease (GERD) is a major risk factor for the development of metaplastic Barrett's esophagus (BE) and its progression to esophageal adenocarcinoma (EAC). Uncontrolled accumulation of reactive oxygen species (ROS) in response to acidic bile salts (ABS) in reflux conditions can be lethal to cells. In this study, we investigated the role of APE1/REF1 in regulating nuclear erythroid factor-like 2 (NRF2), the master antioxidant transcription factor, in response to reflux conditions.

Results: We found that APE1 protein was critical for protecting against cellular ROS levels, oxidative DNA damage, double strand DNA breaks, and cell death in response to conditions that mimic reflux. Analysis of cell lines and de-identified tissues from patients with EAC demonstrated overexpression of both APE1 and NRF2 in EAC cells, as compared to non-neoplastic esophageal cells. Using reflux conditions, we detected concordant and prolonged increases of APE1 and NRF2 protein levels for several hours, following transient short exposure to ABS (20 min). NRF2 transcription activity, as measured by ARE luciferase reporter, and expression of its target genes (HO-1 and TRXND1) were similarly increased in response to ABS. Using genetic knockdown of APE1, we found that APE1 was required for the increase in NRF2 protein stability, nuclear localization, and transcription activation in EAC. Using knockdown of APE1 with reconstitution of wild-type and a redox-deficient mutant (C65A) of APE1, as well as pharmacologic APE1 redox inhibitor (E3330), we demonstrated that APE1 regulated NRF2 in a redox-dependent manner. Mechanistically, we found that APE1 is required for phosphorylation and inactivation of GSK-3β, an important player in the NRF2 degradation pathway.

Conclusion: APE1 redox function was required for ABS-induced activation of NRF2 by regulating phosphorylation and inactivation of GSK-3β. The APE1-NRF2 network played a critical role in protecting esophageal cells against ROS and promoting cell survival under oxidative reflux conditions.
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http://dx.doi.org/10.1016/j.redox.2021.101970DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8082268PMC
July 2021

The development and stem cells of the esophagus.

Development 2021 03 29;148(6). Epub 2021 Mar 29.

Division of Digestive and Liver Disease, Department of Medicine, Columbia University Medical Center, New York, NY 10032, USA

The esophagus is derived from the anterior portion of the foregut endoderm, which also gives rise to the respiratory system. As it develops, the esophageal lining is transformed from a simple columnar epithelium into a stratified squamous cell layer, accompanied by the replacement of unspecified mesenchyme with layers of muscle cells. Studies in animal models have provided significant insights into the roles of various signaling pathways in esophageal development. More recent studies using human pluripotent stem cells (hPSCs) further demonstrate that some of these signaling pathways are conserved in human esophageal development. In addition, a combination of mouse genetics and hPSC differentiation approaches have uncovered new players that control esophageal morphogenesis. In this Review, we summarize these new findings and discuss how the esophagus is established and matures throughout different stages, including its initial specification, respiratory-esophageal separation, epithelial morphogenesis and maintenance. We also discuss esophageal muscular development and enteric nervous system innervation, which are essential for esophageal structure and function.
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http://dx.doi.org/10.1242/dev.193839DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8034879PMC
March 2021

The antioxidant response in Barrett's tumorigenesis: A double-edged sword.

Redox Biol 2021 05 14;41:101894. Epub 2021 Feb 14.

Department of Surgery, University of Miami Miller School of Medicine, Miami, FL, USA; Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, Miami, FL, USA; Department of Veterans Affairs, Miami Healthcare System, Miami, FL, USA. Electronic address:

Esophageal adenocarcinoma (EAC) is the dominant form of esophageal malignancies in the United States and other industrialized countries. The incidence of EAC has been rising rapidly during the past four decades. Barrett's esophagus (BE) is the main precancerous condition for EAC, where a metaplastic columnar epithelium replaces normal squamous mucosa of the lower esophagus. The primary risk factor for BE and EAC are chronic gastroesophageal reflux disease (GERD), obesity and smoking. During the BE-dysplasia-EAC sequence, esophageal cells are under a tremendous burden of accumulating reactive oxygen species (ROS) and oxidative stress. While normal cells have intact antioxidant machinery to maintain a balanced anti-tumorigenic physiological response, the antioxidant capacity is compromised in neoplastic cells with a pro-tumorigenic development antioxidant response. The accumulation of ROS, during the neoplastic progression of the GERD-BE-EAC sequence, induces DNA damage, lipid peroxidation and protein oxidation. Neoplastic cells adapt to oxidative stress by developing a pro-tumorigenic antioxidant response that keeps oxidative damage below lethal levels while promoting tumorigenesis, progression, and resistance to therapy. In this review, we will summarize the recent findings on oxidative stress in tumorigenesis in the context of the GERD-BE-EAC process. We will discuss how EAC cells adapt to increased ROS. We will review APE1 and NRF2 signaling mechanisms in the context of EAC. Finally, we will discuss the potential clinical significance of applying antioxidants or NRF2 activators as chemoprevention and NRF2 inhibitors in treating EAC patients.
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http://dx.doi.org/10.1016/j.redox.2021.101894DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7907897PMC
May 2021

Relationship of the Esophageal Microbiome and Tissue Gene Expression and Links to the Oral Microbiome: A Randomized Clinical Trial.

Clin Transl Gastroenterol 2020 12;11(12):e00235

Department of Medicine, Columbia University Irving Medical Center, New York, New York, USA.

Introduction: Although the microbiome is altered in various esophageal diseases, there is no direct evidence for a link between the oral or esophageal microbiome and underlying esophageal tissue. Here, we aimed to address these gaps through use of an antimicrobial mouth rinse to modify the esophageal microbiome and tissue gene expression.

Methods: In this randomized controlled trial, patients scheduled to undergo endoscopy for clinical indications used chlorhexidine mouth rinse or no treatment for 2 weeks before endoscopy. Oral swabs and saliva were collected at baseline and at follow-up, and the esophagus was sampled on the day of endoscopy. The microbiome was analyzed by 16S rRNA gene sequencing, and esophageal tissue gene expression was ascertained by RNA-Seq.

Results: Twenty subjects were enrolled and included in the analyses. Within individuals, the oral and esophageal microbiome composition was significantly correlated. Chlorhexidine treatment associated with significant alterations to the relative abundance of several esophageal bacterial taxa, and to expression of genes in the esophagus including reductions in periostin, claudin-18, chemokines CXCL1 and CXCL13, and several members of the tumor necrosis factor receptor superfamily. A taxon in genus Haemophilus in the esophagus also associated with significant changes in tissue gene expression.

Discussion: The oral and esophageal microbiomes are closely related within individuals, and esophageal microbiome alterations correlate with tissue gene expression changes. The esophageal microbiome may act as an important cofactor that influences pathogenesis and outcomes of diseases such as eosinophilic esophagitis, gastroesophageal reflux, and Barrett's esophagus.
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http://dx.doi.org/10.14309/ctg.0000000000000235DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7721221PMC
December 2020

Silencing of miR490-3p by H. pylori activates DARPP-32 and induces resistance to gefitinib.

Cancer Lett 2020 10 29;491:87-96. Epub 2020 Jul 29.

Department of Surgery and Department of Pathology, Miler School of Medicine, University of Miami, Miami, FL, USA; Department of Veterans Affairs, Miami Healthcare System, Miami, FL, USA. Electronic address:

Infection with Helicobacter pylori (H. pylori) is the main risk factor for gastric carcinogenesis. In this study, we investigated the expression, molecular functions, and downstream effectors of miR490-3p in gastric cancer. We used in vitro and in vivo models to investigate the role of H. pylori in regulating miR490-3p, DARPP-32-dependent functions, and therapeutic resistance. Human and mouse neoplastic gastric lesions demonstrated a negative correlation between DARPP-32 and miR490-3p expression (R = -0.58, P < 0.01). This was also detected following infection with H. pylori (R = -0.66, P < 0.01). Molecular assays confirmed DARPP-32 as a direct target of miR490-3p. CHRM2, the host gene of miR490-3p, was hypermethylated and downregulated in neoplastic gastric tissues (P < 0.05). H. pylori induced methylation and downregulation of CHRM2 and miR490-3p. Functionally, the reconstitution of miR490-3p sensitized cancer cells to gefitinib by inactivating DRAPP-32-dependent AKT and STAT3 pathways. Patients with low miR490-3p or high DARPP-32 expression had decreased overall survival (P < 0.05). Hypermethylation-mediated silencing of CHRM2 and miR490-3p by H. pylori increased DARPP-32 expression. Downregulation of miR490-3p in gastric cancer plays a role in gefitinib response by inducing DARPP-32-mediated activation of PI3K/AKT, STAT3 signaling pathways.
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http://dx.doi.org/10.1016/j.canlet.2020.07.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7541786PMC
October 2020

Novel candidate genes in esophageal atresia/tracheoesophageal fistula identified by exome sequencing.

Eur J Hum Genet 2021 01 8;29(1):122-130. Epub 2020 Jul 8.

Department of Pediatrics, Columbia University Medical Center, New York, NY, USA.

The various malformations of the aerodigestive tract collectively known as esophageal atresia/tracheoesophageal fistula (EA/TEF) constitute a rare group of birth defects of largely unknown etiology. Previous studies have identified a small number of rare genetic variants causing syndromes associated with EA/TEF. We performed a pilot exome sequencing study of 45 unrelated simplex trios (probands and parents) with EA/TEF. Thirteen had isolated and 32 had nonisolated EA/TEF; none had a family history of EA/TEF. We identified de novo variants in protein-coding regions, including 19 missense variants predicted to be deleterious (D-mis) and 3 likely gene-disrupting (LGD) variants. Consistent with previous studies of structural birth defects, there is a trend of increased burden of de novo D-mis in cases (1.57-fold increase over the background mutation rate), and the burden is greater in constrained genes (2.55-fold, p = 0.003). There is a frameshift de novo variant in EFTUD2, a known EA/TEF risk gene involved in mRNA splicing. Strikingly, 15 out of 19 de novo D-mis variants are located in genes that are putative target genes of EFTUD2 or SOX2 (another known EA/TEF gene), much greater than expected by chance (3.34-fold, p value = 7.20e-5). We estimated that 33% of patients can be attributed to de novo deleterious variants in known and novel genes. We identified APC2, AMER3, PCDH1, GTF3C1, POLR2B, RAB3GAP2, and ITSN1 as plausible candidate genes in the etiology of EA/TEF. We conclude that further genomic analysis to identify de novo variants will likely identify previously undescribed genetic causes of EA/TEF.
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http://dx.doi.org/10.1038/s41431-020-0680-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7852873PMC
January 2021

Distinct stem/progenitor cells proliferate to regenerate the trachea, intrapulmonary airways and alveoli in COVID-19 patients.

Cell Res 2020 08 30;30(8):705-707. Epub 2020 Jun 30.

Center for Human Development and Division of Digestive and Liver Disease, Department of Medicine, Columbia University Medical Center, New York, 10032, NY, USA.

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http://dx.doi.org/10.1038/s41422-020-0367-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7325636PMC
August 2020

Genetic Mouse Models and Induced Pluripotent Stem Cells for Studying Tracheal-Esophageal Separation and Esophageal Development.

Stem Cells Dev 2020 08 2;29(15):953-966. Epub 2020 Jul 2.

Esophageal Development and Engineering Laboratory, Sainte-Justine Research Centre, Montreal, Quebec, Canada.

Esophagus and trachea arise from a common origin, the anterior foregut tube. The compartmentalization process of the foregut into the esophagus and trachea is still poorly understood. Esophageal atresia/tracheoesophageal fistula (EA/TEF) is one of the most common gastrointestinal congenital defects with an incidence rate of 1 in 2,500 births. EA/TEF is linked to the disruption of the compartmentalization process of the foregut tube. In EA/TEF patients, other organ anomalies and disorders have also been reported. Over the last two decades, animal models have shown the involvement of multiple signaling pathways and transcription factors in the development of the esophagus and trachea. Use of induced pluripotent stem cells (iPSCs) to understand organogenesis has been a valuable tool for mimicking gastrointestinal and respiratory organs. This review focuses on the signaling mechanisms involved in esophageal development and the use of iPSCs to model and understand it.
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http://dx.doi.org/10.1089/scd.2020.0075DOI Listing
August 2020

Generation and Characterization of Patient-Derived Head and Neck, Oral, and Esophageal Cancer Organoids.

Curr Protoc Stem Cell Biol 2020 06;53(1):e109

Division of Digestive and Liver Diseases, Department of Medicine and Herbert Irving Comprehensive Cancer Center, Columbia University Irving Medical Center, New York, New York.

Esophageal cancers comprise adenocarcinoma and squamous cell carcinoma, two distinct histologic subtypes. Both are difficult to treat and among the deadliest human malignancies. We describe protocols to initiate, grow, passage, and characterize patient-derived organoids (PDO) of esophageal cancers, as well as squamous cell carcinomas of oral/head-and-neck and anal origin. Formed rapidly (<14 days) from a single-cell suspension embedded in basement membrane matrix, esophageal cancer PDO recapitulate the histology of the original tumors. Additionally, we provide guidelines for morphological analyses and drug testing coupled with functional assessment of cell response to conventional chemotherapeutics and other pharmacological agents in concert with emerging automated imaging platforms. Predicting drug sensitivity and potential therapy resistance mechanisms in a moderate-to-high throughput manner, esophageal cancer PDO are highly translatable in personalized medicine for customized esophageal cancer treatments. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Generation of esophageal cancer PDO Basic Protocol 2: Propagation and cryopreservation of esophageal cancer PDO Basic Protocol 3: Imaged-based monitoring of organoid size and growth kinetics Basic Protocol 4: Harvesting esophageal cancer PDO for histological analyses Basic Protocol 5: PDO content analysis by flow cytometry Basic Protocol 6: Evaluation of drug response with determination of the half-inhibitory concentration (IC ) Support Protocol: Production of RN in HEK293T cell conditioned medium.
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http://dx.doi.org/10.1002/cpsc.109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7350550PMC
June 2020

Inhibition of PU.1 ameliorates metabolic dysfunction and non-alcoholic steatohepatitis.

J Hepatol 2020 08 3;73(2):361-370. Epub 2020 Mar 3.

Naomi Berrie Diabetes Center, Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, 10032, USA. Electronic address:

Background & Aims: Obesity is a well-established risk factor for type 2 diabetes (T2D) and non-alcoholic steatohepatitis (NASH), but the underlying mechanisms remain incompletely understood. Herein, we aimed to identify novel pathogenic factors (and possible therapeutic targets) underlying metabolic dysfunction in the liver.

Methods: We applied a tandem quantitative proteomics strategy to enrich and identify transcription factors (TFs) induced in the obese liver. We used flow cytometry of liver cells to analyze the source of the induced TFs. We employed conditional knockout mice, shRNA, and small-molecule inhibitors to test the metabolic consequences of the induction of identified TFs. Finally, we validated mouse data in patient liver biopsies.

Results: We identified PU.1/SPI1, the master hematopoietic regulator, as one of the most upregulated TFs in livers from diet-induced obese (DIO) and genetically obese (db/db) mice. Targeting PU.1 in the whole liver, but not hepatocytes alone, significantly improved glucose homeostasis and suppressed liver inflammation. Consistently, treatment with the PU.1 inhibitor DB1976 markedly reduced inflammation and improved glucose homeostasis and dyslipidemia in DIO mice, and strongly suppressed glucose intolerance, liver steatosis, inflammation, and fibrosis in a dietary NASH mouse model. Furthermore, hepatic PU.1 expression was positively correlated with insulin resistance and inflammation in liver biopsies from patients.

Conclusions: These data suggest that the elevated hematopoietic factor PU.1 promotes liver metabolic dysfunction, and may be a useful therapeutic target for obesity, insulin resistance/T2D, and NASH.

Lay Summary: Expression of the immune regulator PU.1 is increased in livers of obese mice and people. Blocking PU.1 improved glucose homeostasis, and reduced liver steatosis, inflammation and fibrosis in mouse models of non-alcoholic steatohepatitis. Inhibition of PU.1 is thus a potential therapeutic strategy for treating obesity-associated liver dysfunction and metabolic diseases.
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http://dx.doi.org/10.1016/j.jhep.2020.02.025DOI Listing
August 2020

Targeting SOX2 Protein with Peptide Aptamers for Therapeutic Gains against Esophageal Squamous Cell Carcinoma.

Mol Ther 2020 03 15;28(3):901-913. Epub 2020 Jan 15.

School of Medicine, Xiamen University or Institute for Laboratory Medicine, 900 Hospital of the Joint Logistics Team, Fuzhou, Fujian 350025, China.

Esophageal squamous cell carcinoma (ESCC) is a predominant cancer type in developing countries such as China, where ESCC accounts for approximately 90% of esophageal malignancies. Lacking effective and targeted therapy contributes to the poor 5-year survival rate. Recent studies showed that about 30% of ESCC cases have high levels of SOX2. Herein, we aim to target this transcription factor with aptamer. We established a peptide aptamer library and then performed an unbiased screening to identify several peptide aptamers including P42 that can bind and inhibit SOX2 downstream target genes. We further found that P42 overexpression or incubation with a synthetic peptide 42 inhibited the proliferation, migration, and invasion of ESCC cells. Moreover, peptide 42 treatment inhibited the growth and metastasis of ESCC xenografts in mouse and zebrafish. Further analysis revealed that P42 overexpression led to alternations in the levels of proteins that are important for the proliferation and migration of ESCC cells. Taken together, our study identified the peptide 42 as a key inhibitor of SOX2 function, reducing the proliferation and migration of ESCC cells in vitro and in vivo, and thereby offering a potential therapy against ESCC.
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http://dx.doi.org/10.1016/j.ymthe.2020.01.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7054732PMC
March 2020

Isl1 Regulation of Nkx2.1 in the Early Foregut Epithelium Is Required for Trachea-Esophageal Separation and Lung Lobation.

Dev Cell 2019 12 5;51(6):675-683.e4. Epub 2019 Dec 5.

Division of Digestive and Liver Diseases, Department of Medicine, Columbia University Medical Center, New York, NY 10032, USA; Columbia Center for Human Development, Columbia University Medical Center, New York, NY 10032, USA. Electronic address:

The esophagus and trachea arise from the dorsal and ventral aspects of the anterior foregut, respectively. Abnormal trachea-esophageal separation leads to the common birth defect esophageal atresia with or without trachea-esophageal fistula (EA/TEF). Yet the underlying cellular mechanisms remain unknown. Here, we combine Xenopus and mouse genetic models to identify that the transcription factor Isl1 orchestrates trachea-esophageal separation through modulating a specific epithelial progenitor cell population (midline epithelial cells [MECs], Isl1+ Nkx2.1+ Sox2+) located at the dorsal-ventral boundary of the foregut. Lineage tracing experiments show that MECs contribute to both tracheal and esophageal epithelium, and Isl1 is required for Nkx2.1 transcription in MECs. Deletion of the chromosomal region spanning the ISL1 gene has been found in patients with abnormal trachea-esophageal separation. Our studies thus provide definitive evidence that ISL1 is a critical player in the process of foregut morphogenesis, acting in a small progenitor population of boundary cells.
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http://dx.doi.org/10.1016/j.devcel.2019.11.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6919560PMC
December 2019

Use of hPSC-derived 3D organoids and mouse genetics to define the roles of YAP in the development of the esophagus.

Development 2019 12 4;146(23). Epub 2019 Dec 4.

Division of Digestive and Liver Diseases, Department of Medicine, Columbia University Medical Center, New York, NY 10032, USA

Balanced progenitor activities are crucial for the development and maintenance of high turn-over organs such as the esophagus. However, the molecular mechanisms regulating these progenitor activities in the esophagus remain to be elucidated. Here, we demonstrated that Yap is required for the proliferation of esophageal progenitor cells (EPCs) in the developing murine esophagus. We found that Yap deficiency reduces EPC proliferation and stratification whereas persistent Yap activation increases cell proliferation and causes aberrant stratification of the developing esophagus. We further demonstrated that the role of YAP signaling is conserved in the developing human esophagus by utilizing 3D human pluripotent stem cell (hPSC)-derived esophageal organoid culture. Taken together, our studies combining loss/gain-of-function murine models and hPSC differentiation support a key role for YAP in the self-renewal of EPCs and stratification of the esophageal epithelium.
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http://dx.doi.org/10.1242/dev.178855DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6918786PMC
December 2019

Identification of anoctamin 1 (ANO1) as a key driver of esophageal epithelial proliferation in eosinophilic esophagitis.

J Allergy Clin Immunol 2020 01 21;145(1):239-254.e2. Epub 2019 Oct 21.

Division of Allergy and Immunology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio; Mary H Weiser Food Allergy Center and Department of Pathology, Ann Arbor, Mich. Electronic address:

Background: The pathology of eosinophilic esophagitis (EoE) is characterized by eosinophil-rich inflammation, basal zone hyperplasia (BZH), and dilated intercellular spaces, and the underlying processes that drive the pathologic manifestations of the disease remain largely unexplored.

Objective: We sought to investigate the involvement of the calcium-activated chloride channel anoctamin 1 (ANO1) in esophageal proliferation and the histopathologic features of EoE.

Methods: We examined mRNA and protein expression of ANO1 in esophageal biopsy samples from patients with EoE and in mice with EoE. We performed molecular and cellular analyses and ion transport assays on an in vitro esophageal epithelial 3-dimensional model system (EPC2-ALI) and murine models of EoE to define the relationship between expression and function of ANO1 and esophageal epithelial proliferation in patients with EoE.

Results: We observed increased ANO1 expression in esophageal biopsy samples from patients with EoE and in mice with EoE. ANO1 was expressed within the esophageal basal zone, and expression correlated positively with disease severity (eosinophils/high-power field) and BZH. Using an in vitro esophageal epithelial 3-dimensional model system revealed that ANO1 undergoes chromatin modification and rapid upregulation of expression after IL-13 stimulation, that ANO1 is the primary apical IL-13-induced Cl transport mechanism within the esophageal epithelium, and that loss of ANO1-dependent Cl transport abrogated esophageal epithelial proliferation. Mechanistically, ANO1-dependent regulation of basal cell proliferation was associated with modulation of TP63 expression and phosphorylated cyclin-dependent kinase 2 levels.

Conclusions: These data identify a functional role for ANO1 in esophageal cell proliferation and BZH in patients with EoE and provide a rationale for pharmacologic intervention of ANO1 function in patients with EoE.
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http://dx.doi.org/10.1016/j.jaci.2019.07.049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7366251PMC
January 2020

BMP Signaling in Development, Stem Cells, and Diseases of the Gastrointestinal Tract.

Annu Rev Physiol 2020 02 16;82:251-273. Epub 2019 Oct 16.

Division of Digestive and Liver Diseases, Department of Medicine, Columbia University, New York, NY 10032, USA; email:

The bone morphogenetic protein (BMP) pathway is essential for the morphogenesis of multiple organs in the digestive system. Abnormal BMP signaling has also been associated with disease initiation and progression in the gastrointestinal (GI) tract and associated organs. Recent studies using animal models, tissue organoids, and human pluripotent stem cells have significantly expanded our understanding of the roles played by BMPs in the development and homeostasis of GI organs. It is clear that BMP signaling regulates GI function and disease progression that involve stem/progenitor cells and inflammation in a tissue-specific manner. In this review we discuss these new findings with a focus on the esophagus, stomach, and intestine.
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http://dx.doi.org/10.1146/annurev-physiol-021119-034500DOI Listing
February 2020

Chromatin Assembly Factor 1 (CAF-1) facilitates the establishment of facultative heterochromatin during pluripotency exit.

Nucleic Acids Res 2019 12;47(21):11114-11131

Institute for Cancer Genetics, Columbia University, New York, NY 10032, USA.

Establishment and subsequent maintenance of distinct chromatin domains during embryonic stem cell (ESC) differentiation are crucial for lineage specification and cell fate determination. Here we show that the histone chaperone Chromatin Assembly Factor 1 (CAF-1), which is recruited to DNA replication forks through its interaction with proliferating cell nuclear antigen (PCNA) for nucleosome assembly, participates in the establishment of H3K27me3-mediated silencing during differentiation. Deletion of CAF-1 p150 subunit impairs the silencing of many genes including Oct4, Sox2 and Nanog as well as the establishment of H3K27me3 at these gene promoters during ESC differentiation. Mutations of PCNA residues involved in recruiting CAF-1 to the chromatin also result in defects in differentiation in vitro and impair early embryonic development as p150 deletion. Together, these results reveal that the CAF-1-PCNA nucleosome assembly pathway plays an important role in the establishment of H3K27me3-mediated silencing during cell fate determination.
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http://dx.doi.org/10.1093/nar/gkz858DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6868363PMC
December 2019

Diversified Application of Barcoded PLATO (PLATO-BC) Platform for Identification of Protein Interactions.

Genomics Proteomics Bioinformatics 2019 06 5;17(3):319-331. Epub 2019 Sep 5.

Department of Pathology, Ohio State University Wexner Medical Center, Columbus, OH 43210, USA. Electronic address:

Proteins usually associate with other molecules physically to execute their functions. Identifying these interactions is important for the functional analysis of proteins. Previously, we reported the parallel analysis of translated ORFs (PLATO) to couple ribosome display of full-length ORFs with affinity enrichment of mRNA/protein/ribosome complexes for the "bait" molecules, followed by the deep sequencing analysis of mRNA. However, the sample processing, from extraction of precipitated mRNA to generation of DNA libraries, includes numerous steps, which is tedious and may cause the loss of materials. Barcoded PLATO (PLATO-BC), an improved platform was further developed to test its application for protein interaction discovery. In this report, we tested the antisera-antigen interaction using serum samples from patients with inclusion body myositis (IBM). Tripartite motif containing 21 (TRIM21) was identified as a potentially new IBM autoantigen. We also expanded the application of PLATO-BC to identify protein interactions for JQ1, single ubiquitin peptide, and NS5 protein of Zika virus. From PLATO-BC analyses, we identified new protein interactions for these "bait" molecules. We demonstrate that Ewing sarcoma breakpoint region 1 (EWSR1) binds to JQ1 and their interactions may interrupt the EWSR1 association with acetylated histone H4. RIO kinase 3 (RIOK3), a newly identified ubiquitin-binding protein, is preferentially associated with K63-ubiquitin chain. We also find that Zika NS5 protein interacts with two previously unreported host proteins, par-3 family cell polarity regulator (PARD3) and chromosome 19 open reading frame 53 (C19orf53), whose attenuated expression benefits the replication of Zika virus. These results further demonstrate that PLATO-BC is capable of identifying novel protein interactions for various types of "bait" molecules.
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http://dx.doi.org/10.1016/j.gpb.2018.12.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6818353PMC
June 2019

A CRISPR/Cas9 screen identifies the histone demethylase MINA53 as a novel HIV-1 latency-promoting gene (LPG).

Nucleic Acids Res 2019 08;47(14):7333-7347

Department of Pathology, The Ohio State University Wexner Medical Center, Columbus, OH 43210, USA.

Although combination antiretroviral therapy is potent to block active replication of HIV-1 in AIDS patients, HIV-1 persists as transcriptionally inactive proviruses in infected cells. These HIV-1 latent reservoirs remain a major obstacle for clearance of HIV-1. Investigation of host factors regulating HIV-1 latency is critical for developing novel antiretroviral reagents to eliminate HIV-1 latent reservoirs. From our recently accomplished CRISPR/Cas9 sgRNA screens, we identified that the histone demethylase, MINA53, is potentially a novel HIV-1 latency-promoting gene (LPG). We next validated MINA53's function in maintenance of HIV-1 latency by depleting MINA53 using the alternative RNAi approach. We further identified that in vitro MINA53 preferentially demethylates the histone substrate, H3K36me3 and that in cells MINA53 depletion by RNAi also increases the local level of H3K36me3 at LTR. The effort to map the downstream effectors unraveled that H3K36me3 has the cross-talk with another epigenetic mark H4K16ac, mediated by KAT8 that recognizes the methylated H3K36 and acetylated H4K16. Removing the MINA53-mediated latency mechanisms could benefit the reversal of post-integrated latent HIV-1 proviruses for purging of reservoir cells. We further demonstrated that a pan jumonji histone demethylase inhibitor, JIB-04, inhibits MINA53-mediated demethylation of H3K36me3, and JIB-04 synergizes with other latency-reversing agents (LRAs) to reactivate latent HIV-1.
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http://dx.doi.org/10.1093/nar/gkz493DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6698651PMC
August 2019
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