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1.
Cell ; 186(8): 1523-1527, 2023 04 13.
Article in English | MEDLINE | ID: mdl-37059060

ABSTRACT

Our understanding of tumorigenesis and cancer progression as well as clinical therapies for different cancer types have evolved dramatically in recent years. However, even with this progress, there are big challenges for scientists and oncologists to tackle, ranging from unpacking the molecular and cellular mechanisms involved to therapeutics and biomarker development to quality of life in the aftermath of therapy. In this article, we asked researchers to comment on the questions that they think are important to address in the coming years.


Subject(s)
Neoplasms , Research Personnel , Humans , Carcinogenesis , Neoplasms/blood , Neoplasms/pathology , Neoplasms/therapy , Quality of Life , Research , Biomarkers, Tumor/blood
2.
J Pathol ; 262(3): 362-376, 2024 03.
Article in English | MEDLINE | ID: mdl-38229586

ABSTRACT

Gastroenteropancreatic neuroendocrine tumors (GEP-NETs) are heterogeneous malignancies that arise from complex cellular interactions within the tissue microenvironment. Here, we sought to decipher tumor-derived signals from the surrounding microenvironment by applying digital spatial profiling (DSP) to hormone-secreting and non-functional GEP-NETs. By combining this approach with in vitro studies of human-derived organoids, we demonstrated the convergence of cell autonomous immune and pro-inflammatory proteins that suggests their role in neuroendocrine differentiation and tumorigenesis. DSP was used to evaluate the expression of 40 neural- and immune-related proteins in surgically resected duodenal and pancreatic NETs (n = 20) primarily consisting of gastrinomas (18/20). A total of 279 regions of interest were examined between tumors, adjacent normal and abnormal-appearing epithelium, and the surrounding stroma. The results were stratified by tissue type and multiple endocrine neoplasia I (MEN1) status, whereas protein expression was validated by immunohistochemistry (IHC). A tumor immune cell autonomous inflammatory signature was further evaluated by IHC and RNAscope, while functional pro-inflammatory signaling was confirmed using patient-derived duodenal organoids. Gastrin-secreting and non-functional pancreatic NETs showed a higher abundance of immune cell markers and immune infiltrate compared with duodenal gastrinomas. Compared with non-MEN1 tumors, MEN1 gastrinomas and preneoplastic lesions showed strong immune exclusion and upregulated expression of neuropathological proteins. Despite a paucity of immune cells, duodenal gastrinomas expressed the pro-inflammatory and pro-neural factor IL-17B. Treatment of human duodenal organoids with IL-17B activated NF-κB and STAT3 signaling and induced the expression of neuroendocrine markers. In conclusion, multiplexed spatial protein analysis identified tissue-specific neuro-immune signatures in GEP-NETs. Duodenal gastrinomas are characterized by an immunologically cold microenvironment that permits cellular reprogramming and neoplastic transformation of the preneoplastic epithelium. Moreover, duodenal gastrinomas cell autonomously express immune and pro-inflammatory factors, including tumor-derived IL-17B, that stimulate the neuroendocrine phenotype. © 2024 The Pathological Society of Great Britain and Ireland.


Subject(s)
Duodenal Neoplasms , Gastrinoma , Intestinal Neoplasms , Neuroendocrine Tumors , Pancreatic Neoplasms , Stomach Neoplasms , Humans , Neuroendocrine Tumors/pathology , Gastrinoma/genetics , Gastrinoma/metabolism , Gastrinoma/pathology , Neuroimmunomodulation , Interleukin-17 , Duodenal Neoplasms/genetics , Pancreatic Neoplasms/pathology , Tumor Microenvironment
3.
FASEB J ; 37(5): e22913, 2023 05.
Article in English | MEDLINE | ID: mdl-37078545

ABSTRACT

The Multiple Endocrine Neoplasia I (MEN1) locus encodes the protein MENIN, which functions as a tumor suppressor protein in neuroendocrine tissues. Gastrinomas are neuroendocrine neoplasms that overproduce the hormone gastrin and can arise sporadically or as part of the MEN1 syndrome, in which mutations in the MEN1 gene lead to loss or inactivation of MENIN protein. Gastrin is a peptide hormone that is primarily synthesized in the gastric antrum and stimulates the secretion of histamine from enterochromaffin-like (ECL) cells and subsequently acid from parietal cells in the gastric corpus. In addition, gastrin exerts a mitogenic function primarily on ECL cells and progenitor cells in the gastric isthmus. Current studies seek to understand how MEN1 mutations generate a mutant MENIN protein that abrogates its tumor suppressor function. Mutations in the MEN1 gene are broadly distributed throughout its nine protein-coding exons, making it difficult to correlate protein structure with its function. Although disruption of the Men1 locus in mice causes functional neuroendocrine tumors in the pituitary and pancreas, gastrinomas do not develop in these transgenic animal models. Prior studies of human gastrinomas suggest that tissue-specific microenvironmental cues in the submucosal foregut may contribute to tumorigenesis by reprogramming of epithelial cells toward the neuroendocrine phenotype. Accordingly, recent studies suggest that neural crest-derived cells are also sensitive to reprogramming when MEN1 is deleted or mutated. Thus, the goal of this report is to review our current understanding of how MENIN modulates gastrin gene expression while highlighting its role in the prevention/suppression of neuroendocrine cell transformation.


Subject(s)
Gastrinoma , Multiple Endocrine Neoplasia Type 1 , Pancreatic Neoplasms , Humans , Animals , Mice , Gastrinoma/genetics , Gastrinoma/pathology , Gastrins/genetics , Gastrins/metabolism , Multiple Endocrine Neoplasia Type 1/genetics , Multiple Endocrine Neoplasia Type 1/metabolism , Transcription Factors/genetics , Pancreatic Neoplasms/pathology , Gene Expression , Proto-Oncogene Proteins/genetics
4.
Int J Mol Sci ; 25(5)2024 Mar 04.
Article in English | MEDLINE | ID: mdl-38474232

ABSTRACT

Gastrointestinal cancers represent one of the more challenging cancers to treat. Current strategies to cure and control gastrointestinal (GI) cancers like surgery, radiation, chemotherapy, and immunotherapy have met with limited success, and research has turned towards further characterizing the tumor microenvironment to develop novel therapeutics. Myeloid-derived suppressor cells (MDSCs) have emerged as crucial drivers of pathogenesis and progression within the tumor microenvironment in GI malignancies. Many MDSCs clinical targets have been defined in preclinical models, that potentially play an integral role in blocking recruitment and expansion, promoting MDSC differentiation into mature myeloid cells, depleting existing MDSCs, altering MDSC metabolic pathways, and directly inhibiting MDSC function. This review article analyzes the role of MDSCs in GI cancers as viable therapeutic targets for gastrointestinal malignancies and reviews the existing clinical trial landscape of recently completed and ongoing clinical studies testing novel therapeutics in GI cancers.


Subject(s)
Gastrointestinal Neoplasms , Myeloid-Derived Suppressor Cells , Humans , Myeloid-Derived Suppressor Cells/metabolism , Gastrointestinal Neoplasms/metabolism , Myeloid Cells , Immunotherapy , Tumor Microenvironment
5.
Am J Physiol Gastrointest Liver Physiol ; 325(5): G458-G470, 2023 11 01.
Article in English | MEDLINE | ID: mdl-37698169

ABSTRACT

Notch signaling regulates gastrointestinal stem cell proliferation and differentiation yet Notch-regulated transcriptional effectors of gastric epithelial cell differentiation are poorly understood. Here we tested the role of the bHLH transcription factor Achaete-Scute homolog 1 (ASCL1) in gastric epithelial cell differentiation, and its regulation by Notch. Newborn Ascl1 null mice showed a loss of expression of markers of neurogenin-3-dependent enteroendocrine cells, with normal expression of enterochromaffin-like cells, mucous cells, chief cells, and parietal cells. In adult mice, Ascl1 gene expression was observed in the stomach, but not the intestine, with higher expression in antral than corpus epithelium. Lineage tracing in Ascl1-CreERT2; Rosa26-LSL-tdTomato mice revealed single, scattered ASCL1+ cells in the gastric epithelium, demonstrating expression in antral gastrin- and serotonin-producing endocrine cells. ASCL1-expressing endocrine cells persisted for several weeks posttamoxifen labeling with a half-life of approximately 2 months. Lineage tracing in Gastrin-CreERT2 mice demonstrated a similar lifespan for gastrin-producing cells, confirming that gastric endocrine cells are long-lived. Finally, treatment of Ascl1-CreERT2; Rosa26-LSL-tdTomato mice with the pan-Notch inhibitor dibenzazepine increased the number of lineage-labeled cells in the gastric antrum, suggesting that Notch signaling normally inhibits Ascl1 expression. Notch regulation of Ascl1 was also demonstrated in a genetic mouse model of Notch activation, as well as Notch-manipulated antral organoid cultures, thus suggesting that ASCL1 is a key downstream Notch pathway effector promoting endocrine cell differentiation in the gastric epithelium.NEW & NOTEWORTHY Although Notch signaling is known to regulate cellular differentiation in the stomach, downstream effectors are poorly described. Here we demonstrate that the bHLH transcription factor ASCL1 is expressed in endocrine cells in the stomach and is required for formation of neurogenin-3-dependent enteroendocrine cells but not enterochromaffin-like cells. We also demonstrate that Ascl1 expression is inhibited by Notch signaling, suggesting that ASCL1 is a Notch-regulated transcriptional effector directing enteroendocrine cell fate in the mouse stomach.


Subject(s)
Gastrins , Stomach , Animals , Mice , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Cell Differentiation/physiology , Enteroendocrine Cells/metabolism , Mice, Knockout
6.
Am J Physiol Gastrointest Liver Physiol ; 324(3): G177-G189, 2023 03 01.
Article in English | MEDLINE | ID: mdl-36537709

ABSTRACT

Small intestinal neuroendocrine tumors (SI-NETs) are serotonin-secreting well-differentiated neuroendocrine tumors of putative enterochromaffin (EC) cell origin. However, EC cell-derived tumorigenesis remains poorly understood. Here, we examined whether the gain of Myc and the loss of RB1 and Trp53 function in EC cells result in SI-NET using tryptophan hydroxylase 1 (TPH1) Cre-ERT2-driven RB1fl Trp53fl MycLSL (RPM) mice. TPH1-Cre-induced gain of Myc and loss of RB1 and Trp53 function resulted in endocrine or neuronal tumors in pancreas, lung, enteric neurons, and brain. Lineage tracing indicated that the cellular origin for these tumors was TPH1-expressing neuroendocrine, neuronal, or their precursor cells in these organs. However, despite that TPH1 is most highly expressed in EC cells of the small intestine, we observed no incidence of EC cell tumors. Instead, the tumor of epithelial cell origin in the intestine was exclusively nonendocrine adenocarcinoma, suggesting dedifferentiation of EC cells into intestinal stem cells (ISCs) as a cellular mechanism. Furthermore, ex vivo organoid studies indicated that loss of functions of Rb1 and Trp53 accelerated dedifferentiation of EC cells that were susceptible to apoptosis with expression of activated MycT58A, suggesting that the rare dedifferentiating cells escaping cell death went on to develop adenocarcinomas. Lineage tracing demonstrated that EC cells in the small intestine were short-lived compared with neuroendocrine or neuronal cells in other organs. In contrast, EC cell-derived ISCs were long-lasting and actively cycling and thus susceptible to transformation. These results suggest that tissue- and cell-specific properties of EC cells such as rapid cell turnover and homeostatic dedifferentiation, affect the fate and rate of tumorigenesis induced by genetic alterations and provide important insights into EC cell-derived tumorigenesis.NEW & NOTEWORTHY Small intestinal neuroendocrine tumors are of putative enterochromaffin (EC) cell origin and are the most common malignancy in the small intestine, followed by adenocarcinoma. However, the tumorigenesis of these tumor types remains poorly understood. The present lineage tracing studies showed that tissue- and cell-specific properties of EC cells such as rapid cell turnover and homeostatic dedifferentiation affect the fate and rate of tumorigenesis induced by genetic alterations toward a rare occurrence of adenocarcinoma.


Subject(s)
Adenocarcinoma , Intestinal Neoplasms , Neuroendocrine Tumors , Mice , Animals , Enterochromaffin Cells/metabolism , Intestine, Small/pathology , Carcinogenesis/metabolism , Cell Transformation, Neoplastic/metabolism , Intestinal Neoplasms/metabolism , Neuroendocrine Tumors/metabolism , Adenocarcinoma/genetics
7.
Gastroenterology ; 163(2): 411-425.e4, 2022 08.
Article in English | MEDLINE | ID: mdl-35487288

ABSTRACT

BACKGROUND & AIMS: A subset of myeloid-derived suppressor cells (MDSCs) that express murine Schlafen4 (SLFN4) or its human ortholog SLFN12L polarize in the Helicobacter-inflamed stomach coincident with intestinal or spasmolytic polypeptide-expressing metaplasia. We propose that individuals with a more robust response to damage-activated molecular patterns and increased Toll-like receptor 9 (TLR9) expression are predisposed to the neoplastic complications of Helicobacter infection. METHODS: A mouse or human Transwell co-culture system composed of dendritic cells (DCs), 2-dimensional gastric epithelial monolayers, and Helicobacter were used to dissect the cellular source of interferon-α (IFNα) in the stomach by flow cytometry. Conditioned media from the co-cultures polarized primary myeloid cells. MDSC activity was determined by T-cell suppression assays. In human subjects with intestinal metaplasia or gastric cancer, the rs5743836 TLR9T>C variant was genotyped and linked to TLR9, IFNα, and SLFN12L expression by immunohistochemistry. Nuclear factor-κB binding to the TLR9 C allele was determined by electrophoretic mobility shift assays. RESULTS: Helicobacter infection induced gastric epithelial and plasmacytoid DC expression of TLR9 and IFNα. Co-culturing primary mouse or human cells with DCs and Helicobacter induced TLR9, IFNα secretion, and SLFN+-MDSC polarization. Neutralizing IFNα in vivo mitigated Helicobacter-induced spasmolytic polypeptide-expressing metaplasia. The TLR9 minor C allele creates a nuclear factor-κB binding site associated with higher levels of TLR9, IFNα, and SLFN12L in Helicobacter-infected stomachs that correlated with a greater incidence of metaplasias and cancer. CONCLUSIONS: TLR9 plays an essential role in the production of IFNα and polarization of SLFN+ MDSCs on Helicobacter infection. Subjects carrying the rs5743836 TLR9 minor C allele are predisposed to neoplastic complications if chronically infected.


Subject(s)
Helicobacter Infections , Myeloid-Derived Suppressor Cells , Stomach Neoplasms , Toll-Like Receptor 9 , Animals , Helicobacter , Helicobacter Infections/genetics , Helicobacter Infections/metabolism , Humans , Interferon-alpha , Metaplasia , Mice , NF-kappa B/metabolism , Stomach Neoplasms/genetics , Stomach Neoplasms/microbiology , Toll-Like Receptor 4 , Toll-Like Receptor 9/genetics , Toll-Like Receptor 9/metabolism
8.
Helicobacter ; 28(2): e12954, 2023 Apr.
Article in English | MEDLINE | ID: mdl-36748455

ABSTRACT

BACKGROUND: Helicobacter pylori (Hp) is among the most common bacterial infections in the world and one of the most common infectious agents linked to malignancy, gastric cancer (GC). Within the US there is high disparity in the rates of Hp infection and associated diseases. Hp infection is treatable, and knowledge may influence screening and treatment seeking behaviors. MATERIALS AND METHODS: In this cross-sectional study of 1042 respondents recruited from the Online Amazon MTurk platform, we sought to assess baseline knowledge of Hp and to gain insight into barriers related to Hp care. RESULTS: Just over half (52.3%) reported some prior knowledge of Hp with 11.7% (n = 122) reporting being treated for Hp themselves and 21.4% reporting family members diagnosed with Hp. Of respondents reporting prior treatment, 95 (78%) reported GI upset and 27 (21%) reported not completing medications. Specific to Hp and GC, 70% indicated that a belief that the treatment was worse than the symptoms would affect their willingness to seek care, while 81% indicated knowing Hp can cause GC would affect their treatment decisions and knowing their gastric symptoms were caused by Hp would affect their willingness to receive care. CONCLUSIONS: Knowledge of Hp in this US sample of online respondents is low and self-reported difficulties with treatment compliance is high. Increasing awareness of this infection and addressing the challenges to treatment compliance could potentially reduce rates of Hp antibiotic resistance and progression to GC or other complications of Hp infection.


Subject(s)
Helicobacter Infections , Helicobacter pylori , Stomach Neoplasms , Humans , Helicobacter Infections/drug therapy , Cross-Sectional Studies , Stomach Neoplasms/microbiology
9.
Lasers Surg Med ; 55(2): 208-225, 2023 02.
Article in English | MEDLINE | ID: mdl-36515355

ABSTRACT

BACKGROUND: Duodenal gastrinomas (DGASTs) are neuroendocrine tumors that develop in the submucosa of the duodenum and produce the hormone gastrin. Surgical resection of DGASTs is complicated by the small size of these tumors and the tendency for them to develop diffusely in the duodenum. Endoscopic mucosal resection of DGASTs is an increasingly popular method for treating this disease due to its low complication rate but suffers from poor rates of pathologically negative margins. Multiphoton microscopy can capture high-resolution images of biological tissue with contrast generated from endogenous fluorescence (autofluorescence [AF]) through two-photon excited fluorescence (2PEF). Second harmonic generation is another popular method of generating image contrast with multiphoton microscopy (MPM) and is a light-scattering phenomenon that occurs predominantly from structures such as collagen in biological samples. Some molecules that contribute to AF change in abundance from processes related to the cancer disease process (e.g., metabolic changes, oxidative stress, and angiogenesis). STUDY DESIGN/MATERIALS AND METHODS: MPM was used to image 12 separate patient samples of formalin-fixed and paraffin-embedded duodenal gastrinoma slides with a second-harmonic generation (SHG) channel and four 2PEF channels. The excitation and emission profiles of each 2PEF channel were tuned to capture signal dominated by distinct fluorophores with well-characterized fluorescent spectra and known connections to the physiologic changes that arise in cancerous tissue. RESULTS: We found that there was a significant difference in the relative abundance of signal generated in the 2PEF channels for regions of DGASTs in comparison to the neighboring tissues of the duodenum. Data generated from texture feature extraction of the MPM images were used in linear discriminant analysis models to create classifiers for tumor versus all other tissue types before and after principal component analysis (PCA). PCA improved the classifier accuracy and reduced the number of features required to achieve maximum accuracy. The linear discriminant classifier after PCA distinguished between tumor and other tissue types with an accuracy of 90.6%-93.8%. CONCLUSIONS: These results suggest that multiphoton microscopy 2PEF and SHG imaging is a promising label-free method for discriminating between DGASTs and normal duodenal tissue which has implications for future applications of in vivo assessment of resection margins with endoscopic MPM.


Subject(s)
Gastrinoma , Pancreatic Neoplasms , Humans , Gastrinoma/diagnostic imaging , Gastrinoma/surgery , Microscopy , Endoscopy , Margins of Excision , Pancreatic Neoplasms/diagnostic imaging , Pancreatic Neoplasms/surgery , Microscopy, Fluorescence, Multiphoton/methods
10.
Gut ; 69(10): 1750-1761, 2020 10.
Article in English | MEDLINE | ID: mdl-31980446

ABSTRACT

The myeloid differentiation factor Schlafen4 (Slfn4) marks a subset of myeloid-derived suppressor cells (MDSCs) in the stomach during Helicobacter-induced spasmolytic polypeptide-expressing metaplasia (SPEM). OBJECTIVE: To identify the gene products expressed by Slfn4+-MDSCs and to determine how they promote SPEM. DESIGN: We performed transcriptome analyses for both coding genes (mRNA by RNA-Seq) and non-coding genes (microRNAs using NanoString nCounter) using flow-sorted SLFN4+ and SLFN4- cells from Helicobacter-infected mice exhibiting metaplasia at 6 months postinfection. Thioglycollate-elicited myeloid cells from the peritoneum were cultured and treated with IFNα to induce the T cell suppressor phenotype, expression of MIR130b and SLFN4. MIR130b expression in human gastric tissue including gastric cancer and patient sera was determined by qPCR and in situ hybridisation. Knockdown of MiR130b in vivo in Helicobacter-infected mice was performed using Invivofectamine. Organoids from primary gastric cancers were used to generate xenografts. ChIP assay and Western blots were performed to demonstrate NFκb p65 activation by MIR130b. RESULTS: MicroRNA analysis identified an increase in MiR130b in gastric SLFN4+ cells. Moreover, MIR130b colocalised with SLFN12L, a human homologue of SLFN4, in gastric cancers. MiR130b was required for the T-cell suppressor phenotype exhibited by the SLFN4+ cells and promoted Helicobacter-induced metaplasia. Treating gastric organoids with the MIR130b mimic induced epithelial cell proliferation and promoted xenograft tumour growth. CONCLUSION: Taken together, MiR130b plays an essential role in MDSC function and supports metaplastic transformation.


Subject(s)
Carrier Proteins/metabolism , Helicobacter Infections , MicroRNAs/metabolism , Stomach Neoplasms , Animals , Cell Transformation, Neoplastic/genetics , Disease Models, Animal , Gene Expression Regulation, Neoplastic , Helicobacter Infections/metabolism , Helicobacter Infections/pathology , Helicobacter pylori/physiology , Interferon-alpha/metabolism , Mice , Mice, Knockout , Myeloid-Derived Suppressor Cells/metabolism , Precancerous Conditions , Stomach Neoplasms/metabolism , Stomach Neoplasms/microbiology , Stomach Neoplasms/pathology
11.
Arterioscler Thromb Vasc Biol ; 39(1): 73-88, 2019 01.
Article in English | MEDLINE | ID: mdl-30580567

ABSTRACT

Objective- The goal of this study was to determine the role of ZFP148 (zinc-finger protein 148) in aneurysm formation. Approach and Results- ZFP148 mRNA expression increased at day 3, 7, 14, 21, and 28 after during abdominal aortic aneurysm formation in C57BL/6 mice. Loss of ZFP148 conferred abdominal aortic aneurysm protection using ERTCre+ ZFP148 flx/flx mice. In a third set of experiments, smooth muscle-specific loss of ZFP148 alleles resulted in progressively greater protection using novel transgenic mice (MYH [myosin heavy chain 11] Cre+ flx/flx, flx/wt, and wt/wt). Elastin degradation, LGAL3, and neutrophil staining were significantly attenuated, while α-actin staining was increased in ZFP148 knockout mice. Results were verified in total cell ZFP148 and smooth muscle-specific knockout mice using an angiotensin II model. ZFP148 smooth muscle-specific conditional mice demonstrated increased proliferation and ZFP148 was shown to bind to the p21 promoter during abdominal aortic aneurysm formation. ZFP148 smooth muscle-specific conditional knockout mice also demonstrated decreased apoptosis as measured by decreased cleaved caspase-3 staining. ZFP148 bound smooth muscle marker genes via chromatin immunoprecipitation analysis mediated by NF-1 (neurofibromin 1) promote histone H3K4 deacetylation via histone deacetylase 5. Transient transfections and chromatin immunoprecipitation analyses demonstrated that NF-1 was required for ZFP148 protein binding to smooth muscle marker genes promoters during aneurysm formation. Elimination of NF-1 using shRNA approaches demonstrated that NF-1 is required for binding and elimination of NF-1 increased BRG1 recruitment, the ATPase subunit of the SWI/SWF complex, and increased histone acetylation. Conclusions- ZFP148 plays a critical role in multiple murine models of aneurysm formation. These results suggest that ZFP148 is important in the regulation of proliferation, smooth muscle gene downregulation, and apoptosis in aneurysm development.


Subject(s)
Aortic Aneurysm, Abdominal/etiology , DNA-Binding Proteins/metabolism , Myocytes, Smooth Muscle/metabolism , Neurofibromin 1/metabolism , Transcription Factors/metabolism , Acetylation , Angiotensin II/pharmacology , Animals , Aortic Aneurysm, Abdominal/metabolism , Apoptosis , Cell Proliferation , Cyclin-Dependent Kinase Inhibitor p21/genetics , Female , Histones/metabolism , Humans , Male , Mice , Mice, Inbred C57BL , bcl-2 Homologous Antagonist-Killer Protein/genetics
12.
Am J Physiol Gastrointest Liver Physiol ; 315(4): G495-G510, 2018 10 01.
Article in English | MEDLINE | ID: mdl-29848020

ABSTRACT

Lgr5-expressing intestinal stem cells (ISCs) maintain continuous and rapid generation of the intestinal epithelium. Here, we present evidence that dedifferentiation of committed enteroendocrine cells (EECs) contributes to maintenance of the epithelium under both basal conditions and in response to injury. Lineage-tracing studies identified a subset of EECs that reside at +4 position for more than 2 wk, most of which were BrdU-label-retaining cells. Under basal conditions, cells derived from these EECs grow from the bottom of the crypt to generate intestinal epithelium according to neutral drift kinetics that is consistent with dedifferentiation of mature EECs to ISCs. The lineage tracing of EECs demonstrated reserve stem cell properties in response to radiation-induced injury with the generation of reparative EEC-derived epithelial patches. Finally, the enterochromaffin (EC) cell was the predominant EEC type participating in these stem cell dynamics. These results provide novel insights into the +4 reserve ISC hypothesis, stem cell dynamics of the intestinal epithelium, and in the development of EC-derived small intestinal tumors. NEW & NOTEWORTHY The current manuscript demonstrating that a subset of mature enteroendocrine cells (EECs), predominantly enterochromaffin cells, dedifferentiates to fully functional intestinal stem cells (ISCs) is novel, timely, and important. These cells dedifferentiate to ISCs not only in response to injury but also under basal homeostatic conditions. These novel findings provide a mechanism in which a specified cell can dedifferentiate and contribute to normal tissue plasticity as well as the development of EEC-derived intestinal tumors under pathologic conditions.


Subject(s)
Adult Stem Cells/cytology , Cell Differentiation , Cell Proliferation , Enteroendocrine Cells/cytology , Intestine, Small/cytology , Adult Stem Cells/metabolism , Animals , Cells, Cultured , Enteroendocrine Cells/metabolism , Intestine, Small/pathology , Mice , Mice, Inbred C57BL , Radiation Injuries, Experimental/pathology , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism
13.
Gastroenterology ; 153(6): 1555-1567.e15, 2017 12.
Article in English | MEDLINE | ID: mdl-28859856

ABSTRACT

BACKGROUND & AIMS: The multiple endocrine neoplasia, type 1 (MEN1) locus encodes the nuclear protein and tumor suppressor menin. MEN1 mutations frequently cause neuroendocrine tumors such as gastrinomas, characterized by their predominant duodenal location and local metastasis at time of diagnosis. Diffuse gastrin cell hyperplasia precedes the appearance of MEN1 gastrinomas, which develop within submucosal Brunner's glands. We investigated how menin regulates expression of the gastrin gene and induces generation of submucosal gastrin-expressing cell hyperplasia. METHODS: Primary enteric glial cultures were generated from the VillinCre:Men1FL/FL:Sst-/- mice or C57BL/6 mice (controls), with or without inhibition of gastric acid by omeprazole. Primary enteric glial cells from C57BL/6 mice were incubated with gastrin and separated into nuclear and cytoplasmic fractions. Cells were incubated with forskolin and H89 to activate or inhibit protein kinase A (a family of enzymes whose activity depends on cellular levels of cyclic AMP). Gastrin was measured in blood, tissue, and cell cultures using an ELISA. Immunoprecipitation with menin or ubiquitin was used to demonstrate post-translational modification of menin. Primary glial cells were incubated with leptomycin b and MG132 to block nuclear export and proteasome activity, respectively. We obtained human duodenal, lymph node, and pancreatic gastrinoma samples, collected from patients who underwent surgery from 1996 through 2007 in the United States or the United Kingdom. RESULTS: Enteric glial cells that stained positive for glial fibrillary acidic protein (GFAP+) expressed gastrin de novo through a mechanism that required PKA. Gastrin-induced nuclear export of menin via cholecystokinin B receptor (CCKBR)-mediated activation of PKA. Once exported from the nucleus, menin was ubiquitinated and degraded by the proteasome. GFAP and other markers of enteric glial cells (eg, p75 and S100B), colocalized with gastrin in human duodenal gastrinomas. CONCLUSIONS: MEN1-associated gastrinomas, which develop in the submucosa, might arise from enteric glial cells through hormone-dependent PKA signaling. This pathway disrupts nuclear menin function, leading to hypergastrinemia and associated sequelae.


Subject(s)
Duodenum/metabolism , Gastrins/metabolism , Neuroglia/enzymology , Proteasome Endopeptidase Complex/metabolism , Proto-Oncogene Proteins/metabolism , Active Transport, Cell Nucleus , Animals , Cells, Cultured , Cyclic AMP-Dependent Protein Kinases/metabolism , Duodenal Neoplasms/enzymology , Duodenal Neoplasms/genetics , Duodenal Neoplasms/pathology , Duodenum/drug effects , Duodenum/pathology , Gastrinoma/enzymology , Gastrinoma/genetics , Gastrinoma/pathology , Gastrins/genetics , Gene Expression Regulation , Glial Fibrillary Acidic Protein/metabolism , Humans , Hyperplasia , Mice, Inbred C57BL , Mice, Knockout , Neuroglia/drug effects , Proteasome Inhibitors/pharmacology , Proteolysis , Proto-Oncogene Proteins/genetics , Proton Pump Inhibitors/pharmacology , Receptor, Cholecystokinin B/metabolism , Receptors, Somatostatin/genetics , Receptors, Somatostatin/metabolism , Time Factors , Ubiquitination
14.
Nature ; 482(7386): 542-6, 2012 Feb 12.
Article in English | MEDLINE | ID: mdl-22327296

ABSTRACT

Menin is a tumour suppressor protein whose loss or inactivation causes multiple endocrine neoplasia 1 (MEN1), a hereditary autosomal dominant tumour syndrome that is characterized by tumorigenesis in multiple endocrine organs. Menin interacts with many proteins and is involved in a variety of cellular processes. Menin binds the JUN family transcription factor JUND and inhibits its transcriptional activity. Several MEN1 missense mutations disrupt the menin-JUND interaction, suggesting a correlation between the tumour-suppressor function of menin and its suppression of JUND-activated transcription. Menin also interacts with mixed lineage leukaemia protein 1 (MLL1), a histone H3 lysine 4 methyltransferase, and functions as an oncogenic cofactor to upregulate gene transcription and promote MLL1-fusion-protein-induced leukaemogenesis. A recent report on the tethering of MLL1 to chromatin binding factor lens epithelium-derived growth factor (LEDGF) by menin indicates that menin is a molecular adaptor coordinating the functions of multiple proteins. Despite its importance, how menin interacts with many distinct partners and regulates their functions remains poorly understood. Here we present the crystal structures of human menin in its free form and in complexes with MLL1 or with JUND, or with an MLL1-LEDGF heterodimer. These structures show that menin contains a deep pocket that binds short peptides of MLL1 or JUND in the same manner, but that it can have opposite effects on transcription. The menin-JUND interaction blocks JUN N-terminal kinase (JNK)-mediated JUND phosphorylation and suppresses JUND-induced transcription. In contrast, menin promotes gene transcription by binding the transcription activator MLL1 through the peptide pocket while still interacting with the chromatin-anchoring protein LEDGF at a distinct surface formed by both menin and MLL1.


Subject(s)
Myeloid-Lymphoid Leukemia Protein/metabolism , Proto-Oncogene Proteins c-jun/metabolism , Proto-Oncogene Proteins/chemistry , Proto-Oncogene Proteins/metabolism , Transcription, Genetic , Amino Acid Motifs , Amino Acid Sequence , Animals , Binding Sites , Chromatin/metabolism , Crystallography, X-Ray , Fibroblasts , HEK293 Cells , Histone-Lysine N-Methyltransferase , Humans , Intercellular Signaling Peptides and Proteins/metabolism , JNK Mitogen-Activated Protein Kinases/metabolism , Mice , Models, Molecular , Molecular Sequence Data , Myeloid-Lymphoid Leukemia Protein/chemistry , Phosphorylation , Protein Binding , Protein Multimerization , Proto-Oncogene Proteins c-jun/chemistry , Structure-Activity Relationship
15.
Dig Dis Sci ; 68(1): 16-20, 2023 01.
Article in English | MEDLINE | ID: mdl-36301448
16.
Dig Dis Sci ; 68(1): 21-23, 2023 01.
Article in English | MEDLINE | ID: mdl-36301449

Subject(s)
Mentoring , Humans , Mentors
17.
Gut ; 66(6): 1012-1021, 2017 06.
Article in English | MEDLINE | ID: mdl-26860771

ABSTRACT

BACKGROUND: Gastric carcinoids are slow growing neuroendocrine tumours arising from enterochromaffin-like (ECL) cells in the corpus of stomach. Although most of these tumours arise in the setting of gastric atrophy and hypergastrinemia, it is not understood what genetic background predisposes development of these ECL derived tumours. Moreover, diffuse microcarcinoids in the mucosa can lead to a field effect and limit successful endoscopic removal. OBJECTIVE: To define the genetic background that creates a permissive environment for gastric carcinoids using transgenic mouse lines. DESIGN: The multiple endocrine neoplasia 1 gene locus (Men1) was deleted using Cre recombinase expressed from the Villin promoter (Villin-Cre) and was placed on a somatostatin null genetic background. These transgenic mice received omeprazole-laced chow for 6 months. The direct effect of gastrin and the gastrin receptor antagonist YM022 on expression and phosphorylation of the cyclin inhibitor p27Kip1 was tested on the human human gastric adenocarcinoma cell line stably expressing CCKBR (AGSE) and mouse small intestinal neuroendocrine carcinoma (STC)-1 cell lines. RESULTS: The combination of conditional Men1 deletion in the absence of somatostatin led to the development of gastric carcinoids within 2 years. Suppression of acid secretion by omeprazole accelerated the timeline of carcinoid development to 6 months in the absence of significant parietal cell atrophy. Carcinoids were associated with hypergastrinemia, and correlated with increased Cckbr expression and nuclear export of p27Kip1 both in vivo and in gastrin-treated cell lines. Loss of p27Kip1 was also observed in human gastric carcinoids arising in the setting of atrophic gastritis. CONCLUSIONS: Gastric carcinoids require threshold levels of hypergastrinemia, which modulates p27Kip1 cellular location and stability.


Subject(s)
Adenocarcinoma/metabolism , Carcinoid Tumor/genetics , Gastrins/metabolism , Proto-Oncogene Proteins/genetics , Somatostatin/genetics , Stomach Neoplasms/genetics , Stomach Neoplasms/metabolism , Adult , Animals , Benzodiazepines/pharmacology , Carcinogenesis/drug effects , Carcinogenesis/genetics , Cell Line, Tumor , Cyclin-Dependent Kinase Inhibitor p27/metabolism , Female , Gastrins/blood , Gastrins/genetics , Gastrins/pharmacology , Gene Deletion , Hormone Antagonists/pharmacology , Hormones/pharmacology , Humans , Male , Mice , Mice, Transgenic , Omeprazole/pharmacology , Phosphorylation/drug effects , Proton Pump Inhibitors/pharmacology , RNA, Messenger/metabolism , Receptor, Cholecystokinin B/genetics , Receptor, Cholecystokinin B/metabolism , Signal Transduction
18.
Am J Physiol Gastrointest Liver Physiol ; 312(1): G15-G23, 2017 01 01.
Article in English | MEDLINE | ID: mdl-27810953

ABSTRACT

The impact of omeprazole (OM), a widely used over-the-counter proton pump inhibitor, on weight gain has not been extensively explored. We examined what factors, e.g., diet composition, microbiota, genetic strain, and sex, might affect weight gain in mice fed a high caloric diet while on OM. Inbred C57BL/6J strain, a 50:50 hybrid (B6SJLF1/J) strain, and mice on a highly mixed genetic background were fed four diets: standard chow (STD, 6% fat), STD with 200 ppm OM (STD + O), a high-energy chow (HiE, 11% fat), and HiE chow with OM (HiE + O) for 17 wk. Metabolic analysis, body composition, and fecal microbiota composition were analyzed in C57BL/6J mice. Oral glucose tolerance tests were performed using mice on the mixed background. After 8 wk, female and male C57BL/6J mice on the HiE diets ate less, whereas males on the HiE diets compared with the STD diets gained weight. All diet treatments reduced energy expenditure in females but in males only those on the HiE + O diet. Gut microbiota composition differed in the C57BL/6J females but not the males. Hybrid B6SJLF1/J mice showed similar weight gain on all test diets. In contrast, mixed strain male mice fed a HiE + O diet gained ∼40% more weight than females on the same diet. In addition to increased weight gain, mixed genetic mice on the HiE + O diet cleared glucose normally but secreted more insulin. We concluded that sex and genetic background define weight gain and metabolic responses of mice on high caloric diets and OM.


Subject(s)
Diet, High-Fat , Omeprazole/pharmacology , Proton Pump Inhibitors/pharmacology , Weight Gain/drug effects , Adiposity/drug effects , Adiposity/genetics , Animals , Energy Intake/drug effects , Energy Intake/genetics , Energy Metabolism/drug effects , Energy Metabolism/genetics , Feeding Behavior/drug effects , Feeding Behavior/physiology , Female , Gastrointestinal Microbiome/drug effects , Gastrointestinal Microbiome/genetics , Genetic Background , Male , Mice , Mice, Inbred C57BL , Sex Factors , Weight Gain/genetics
19.
Curr Gastroenterol Rep ; 19(7): 32, 2017 Jul.
Article in English | MEDLINE | ID: mdl-28608155

ABSTRACT

PURPOSE OF REVIEW: Neuroendocrine tumors (NETs) were initially identified as a separate entity in the early 1900s as a unique malignancy that secretes bioactive amines. GI-NETs are the most frequent type and represent a unique subset of NETs, because at least 75% of these tumors represent gastrin stimulation of the enterochromaffin-like cell located in the body of the stomach. The purpose of this review is to understand the specific role of gastrin in the generation of Gastric NETs (G-NETs). RECENT FINDINGS: We review here the origin of enterochromaffin cells gut and the role of hypergastrinemia in gastric enteroendocrine tumorigenesis. We describe generation of the first genetically engineered mouse model of gastrin-driven G-NETs that mimics the human phenotype. The common mechanism observed in both the hypergastrinemic mouse model and human carcinoids is translocation of the cyclin-dependent inhibitor p27kip to the cytoplasm and its subsequent degradation by the proteasome. Therapies that block degradation of p27kip, the CCKBR2 gastrin receptor, or gastrin peptide are likely to facilitate treatment.


Subject(s)
Gastrins/physiology , Neuroendocrine Tumors/etiology , Stomach Neoplasms/etiology , Animals , Carcinoid Tumor/metabolism , Cyclin-Dependent Kinase Inhibitor p27/metabolism , Cytoplasm/metabolism , Disease Models, Animal , Enterochromaffin-like Cells/physiology , Gastrins/blood , Humans , Mice , Neuroendocrine Tumors/metabolism , Phenotype , Receptor, Cholecystokinin B/metabolism , Stomach Neoplasms/metabolism
20.
Gastroenterology ; 149(7): 1849-59, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26261005

ABSTRACT

BACKGROUND & AIMS: Dual oxidase 2 (DUOX2), a hydrogen-peroxide generator at the apical membrane of gastrointestinal epithelia, is up-regulated in patients with inflammatory bowel disease (IBD) before the onset of inflammation, but little is known about its effects. We investigated the role of DUOX2 in maintaining mucosal immune homeostasis in mice. METHODS: We analyzed the regulation of DUOX2 in intestinal tissues of germ-free vs conventional mice, mice given antibiotics or colonized with only segmented filamentous bacteria, mice associated with human microbiota, and mice with deficiencies in interleukin (IL) 23 and IL22 signaling. We performed 16S ribosomal RNA gene quantitative polymerase chain reaction of intestinal mucosa and mesenteric lymph nodes of Duoxa(-/-) mice that lack functional DUOX enzymes. Genes differentially expressed in Duoxa(-/-) mice compared with co-housed wild-type littermates were correlated with gene expression changes in early-stage IBD using gene set enrichment analysis. RESULTS: Colonization of mice with segmented filamentous bacteria up-regulated intestinal expression of DUOX2. DUOX2 regulated redox signaling within mucosa-associated microbes and restricted bacterial access to lymphatic tissues of the mice, thereby reducing microbiota-induced immune responses. Induction of Duox2 transcription by microbial colonization did not require the mucosal cytokines IL17 or IL22, although IL22 increased expression of Duox2. Dysbiotic, but not healthy human microbiota, activated a DUOX2 response in recipient germ-free mice that corresponded to abnormal colonization of the mucosa with distinct populations of microbes. In Duoxa(-/-) mice, abnormalities in ileal mucosal gene expression at homeostasis recapitulated those in patients with mucosal dysbiosis. CONCLUSIONS: DUOX2 regulates interactions between the intestinal microbiota and the mucosa to maintain immune homeostasis in mice. Mucosal dysbiosis leads to increased expression of DUOX2, which might be a marker of perturbed mucosal homeostasis in patients with early-stage IBD.


Subject(s)
Bacteria/pathogenicity , Dysbiosis , Epithelial Cells/microbiology , Gastroenteritis/microbiology , Immunity, Mucosal , Inflammatory Bowel Diseases/microbiology , Intestinal Mucosa/microbiology , NADPH Oxidases/biosynthesis , NADPH Oxidases/metabolism , Salmonella Infections/microbiology , Animals , Anti-Bacterial Agents/pharmacology , Bacteria/classification , Bacteria/drug effects , Bacteria/genetics , Bacteria/immunology , Bacterial Translocation , Disease Models, Animal , Dual Oxidases , Enzyme Induction , Epithelial Cells/drug effects , Epithelial Cells/enzymology , Epithelial Cells/immunology , Feces/microbiology , Female , Gastroenteritis/enzymology , Gastroenteritis/genetics , Gastroenteritis/immunology , Host-Pathogen Interactions , Humans , Inflammatory Bowel Diseases/enzymology , Inflammatory Bowel Diseases/immunology , Interleukins/deficiency , Interleukins/genetics , Intestinal Mucosa/drug effects , Intestinal Mucosa/enzymology , Intestinal Mucosa/immunology , Male , Mice, Inbred C57BL , Mice, Knockout , NADPH Oxidases/deficiency , NADPH Oxidases/genetics , Permeability , Receptors, Interleukin/deficiency , Receptors, Interleukin/genetics , Ribotyping , Salmonella Infections/enzymology , Salmonella Infections/genetics , Salmonella Infections/immunology , Salmonella typhimurium/pathogenicity , Signal Transduction , Tissue Culture Techniques , Transcription, Genetic , Interleukin-22
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