RESUMEN
BACKGROUND & AIMS: CD44s is a surface marker of tumor-initiating cells (TICs); high tumor levels correlate with metastasis and recurrence, as well as poor outcomes for patients. Monoclonal antibodies against CD44s might eliminate TICs with minimal toxicity. This strategy is unclear for treatment of pancreatic cancer, and little is known about how anti-CD44s affect pancreatic cancer initiation or recurrence after radiotherapy. METHODS: One hundred ninety-two pairs of human pancreatic adenocarcinoma and adjacent nontumor pancreatic tissues were collected from patients undergoing surgery. We measured CD44s levels in tissue samples and pancreatic cancer cell lines by immunohistochemistry, real-time polymerase chain reaction, and immunoblot; levels were correlated with patient survival times. We studied the effects of anti-CD44s in mice with human pancreatic tumor xenografts and used flow cytometry to determine the effects on TICs. Changes in CD44s signaling were examined by real-time polymerase chain reaction, immunoblot, reporter assay, and in vitro tumorsphere formation assays. RESULTS: Levels of CD44s were significantly higher in pancreatic cancer than adjacent nontumor tissues. Patients whose tumors expressed high levels of CD44s had a median survival of 10 months compared with >43 months for those with low levels. Anti-CD44s reduced growth, metastasis, and postradiation recurrence of pancreatic xenograft tumors in mice. The antibody reduced the number of TICs in cultured pancreatic cancer cells and xenograft tumors, as well as their tumorigenicity. In cultured pancreatic cancer cell lines, anti-CD44s down-regulated the stem cell self-renewal genes Nanog, Sox-2, and Rex-1 and inhibited signal transducer and activator of transcription 3-mediated cell proliferation and survival signaling. CONCLUSIONS: The TIC marker CD44s is up-regulated in human pancreatic tumors and associated with patient survival time. CD44s is required for initiation, growth, metastasis, and postradiation recurrence of xenograft tumors in mice. Anti-CD44s eliminated bulk tumor cells as well as TICs from the tumors. Strategies to target CD44s cab be developed to block pancreatic tumor formation and post-radiotherapy recurrence in patients.
Asunto(s)
Adenocarcinoma/terapia , Anticuerpos/farmacología , Biomarcadores de Tumor/inmunología , Receptores de Hialuranos/inmunología , Recurrencia Local de Neoplasia/prevención & control , Células Madre Neoplásicas/efectos de los fármacos , Neoplasias Pancreáticas/terapia , Adenocarcinoma/genética , Adenocarcinoma/inmunología , Adenocarcinoma/metabolismo , Adenocarcinoma/mortalidad , Adenocarcinoma/secundario , Animales , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Receptores de Hialuranos/genética , Receptores de Hialuranos/metabolismo , Estimación de Kaplan-Meier , Ratones , Ratones Desnudos , Células Madre Neoplásicas/inmunología , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Células Madre Neoplásicas/efectos de la radiación , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/mortalidad , Neoplasias Pancreáticas/patología , Transducción de Señal/efectos de los fármacos , Esferoides Celulares , Factores de Tiempo , Factores de Transcripción/metabolismo , Carga Tumoral/efectos de los fármacos , Células Tumorales Cultivadas , Regulación hacia Arriba , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Epithelial tuft cells are named after their characteristic microtubule bundles located at the cell apex where these are exposed to the luminal environment. As such, tuft cells are found in multiple organs, including the gastrointestinal (GI) tract where the apical "tuft" is hypothesized to detect and transmit environmental signals. Thus, the goal of our study was to characterize gastric tuft cells during GI tract development, then subsequently in the normal and metaplastic adult stomach. GI tracts from mouse embryos, and newborn and postnatal mice were analyzed. Tuft cells were identified by immunohistochemistry using acetylated-α-tubulin (acTub) antibody to detect the microtubule bundle. Additional tuft cell markers, e.g., doublecortin-like kinase 1 (DCLK1), were used to co-localize with acTub. Tuft cells were quantified in human gastric tissue arrays and in mouse stomachs with or without inflammation. In the developing intestine, tuft cells in both the crypts and villi expressed all markers by E18.5. In the stomach, acTub co-localized with DCLK1 and other established tuft cell markers by E18.5 in the antrum, but not until postnatal day 7 in the corpus, with the highest density of tuft cells clustered at the forestomach ridge. Tuft cell numbers increased in hyperplastic human and mouse stomachs. In the adult GI tract, the tuft cell marker acTub co-expressed with DCKL1 and chemosensory markers, e.g.,TRPM5. In summary, tuft cells appear in the gastric antrum and intestine at E18.5, but their maximal numbers in the corpus are not achieved until after weaning. Tuft cell numbers increase with inflammation, hyperplasia, and metaplasia.
Asunto(s)
Células Quimiorreceptoras/metabolismo , Células Quimiorreceptoras/patología , Mucosa Gástrica/patología , Proteínas Serina-Treonina Quinasas/metabolismo , Antro Pilórico/patología , Animales , Quinasas Similares a Doblecortina , Células Epiteliales/metabolismo , Células Epiteliales/patología , Gastritis/metabolismo , Gastritis/patología , Tracto Gastrointestinal/embriología , Tracto Gastrointestinal/crecimiento & desarrollo , Tracto Gastrointestinal/patología , Humanos , Hiperplasia/metabolismo , Hiperplasia/patología , Inmunohistoquímica , Metaplasia/patología , Ratones , Canales Catiónicos TRPM/metabolismoRESUMEN
In the adult mouse, distinct morphological and transcriptional differences separate stomach from intestinal epithelium. Remarkably, the epithelial boundary between these two organs is literally one cell thick. This discrete junction is established suddenly and precisely at embryonic day (E) 16.5, by sharpening a previously diffuse intermediate zone. In the present study, we define the dynamic transcriptome of stomach, pylorus, and intestinal tissues between E14.5 and E16.5. We show that establishment of this boundary is concomitant with the induction of over a thousand genes in intestinal epithelium, and these gene products provide intestinal character. Hence, we call this process intestinalization. We identify specific transcription factors (Hnf4 gamma, Creb3l3, and Tcfec) and examine signaling pathways (Hedgehog and Wnt) that may play a role in this process. Finally, we define a unique expression domain at the pylorus itself and detect novel pylorus-specific patterns for the transcription factor Gata3 and the secreted protein nephrocan.
Asunto(s)
Desarrollo Fetal/genética , Mucosa Intestinal/embriología , Intestinos/embriología , Píloro/embriología , Píloro/metabolismo , Estómago/embriología , Animales , Embrión de Mamíferos , Femenino , Desarrollo Fetal/fisiología , Mucosa Gástrica/metabolismo , Perfilación de la Expresión Génica , Edad Gestacional , Mucosa Intestinal/metabolismo , Factores de Transcripción de Tipo Kruppel/genética , Operón Lac/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Embarazo , Proteína con Dedos de Zinc GLI1RESUMEN
BACKGROUND & AIMS: Epithelial stem cells in the stomach are responsible for constant renewal of the epithelium through generation of multiple gastric cell lineages that populate the gastric glands. However, gastric stem or progenitor cells have not been well-characterized because of the lack of specific markers that permit their prospective recognition. We identified an intestinal promoter that is active in a rare subpopulation of gastric epithelial cells and investigated whether these cells possess multilineage potential. METHODS: A marked allele of the endogenous mouse villin locus was used to visualize single beta-galactosidase-positive cells located in the lower third of antral glands. A 12.4-kb villin promoter/enhancer fragment drives several transgenes (EGFP, beta-galactosidase, and Cre recombinase) in these cells in a pattern similar to that of the marked villin allele. Reporter gene activity was used to track these cells during development and to examine cell number in the context of inflammatory challenge while Cre activity allowed lineage tracing in vivo. RESULTS: We show that these rare epithelial cells are normally quiescent, but multiply in response to interferon gamma. Lineage tracing studies confirm that these cells give rise to all gastric lineages of the antral glands. In the embryo, these cells are located basally in the stomach epithelium before completion of gastric gland morphogenesis. CONCLUSIONS: We have identified a rare subpopulation of gastric progenitors with multilineage potential. The ability to prospectively identify and manipulate such progenitors in situ represents a major step forward in gastric stem cell biology and has potential implications for gastric cancer.
Asunto(s)
Células Epiteliales/citología , Mucosa Gástrica/citología , Células Madre/citología , Animales , Proliferación Celular/efectos de los fármacos , Interferón gamma/farmacología , Ratones , Ratones Transgénicos , Proteínas de Microfilamentos/genética , Modelos AnimalesRESUMEN
PURPOSE: Signal transducer and activator of transcription 3 (STAT3) plays a critical role in initiation and progression of pancreatic cancer. However, therapeutically targeting STAT3 has failed clinically. We previously identified HAb18G/CD147 as an effective target for cancer treatment. In this study, we aimed to investigate the potential role of HAb18G/CD147 in STAT3-involved pancreatic tumorigenesis in vitro and in vivo. EXPERIMENTAL DESIGN: The expression of HAb18G/CD147, pSTAT3, and CD44s was determined in tissue microarrays. The tumorigenic function and molecular signaling mechanism of HAb18G/CD147 were assessed by in vitro cellular and clonogenic growth, reporter assay, immunoblot assay, immunofluorescence staining, immunoprecipitation, and in vivo tumor formation using loss or gain-of-function strategies. RESULTS: Highly expressed HAb18G/CD147 promoted cellular and clonogenic growth in vitro and tumorigenicity in vivo. Cyclophilin A (CyPA), a ligand of CD147, stimulated STAT3 phosphorylation and its downstream genes cyclin D1/survivin through HAb18G/CD147-dependent mechanisms. HAb18G/CD147 was associated and colocalized with cancer stem cell marker CD44s in lipid rafts. The inhibitors of STAT3 and survivin, as well as CD44s neutralizing antibodies suppressed the HAb18G/CD147-induced cell growth. High HAb18G/CD147 expression in pancreatic cancer was significantly correlated with the poor tumor differentiation, and the high coexpression of HAb18G/CD147-CD44s-STAT3 associated with poor survival of patients with pancreatic cancer. CONCLUSIONS: We identified HAb18G/CD147 as a novel upstream activator of STAT3, which interacts with CD44s and plays a critical role in the development of pancreatic cancer. The data suggest that HAb18G/CD147 could be a promising therapeutic target for highly aggressive pancreatic cancer and a surrogate marker in the STAT3-targeted molecular therapies.
Asunto(s)
Basigina/biosíntesis , Receptores de Hialuranos/biosíntesis , Neoplasias Pancreáticas/genética , Factor de Transcripción STAT3/biosíntesis , Anciano , Animales , Carcinogénesis , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Estimación de Kaplan-Meier , Masculino , Ratones , Persona de Mediana Edad , Neoplasias Pancreáticas/patología , Transducción de Señal/genética , Análisis de Matrices Tisulares , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Gastric stem and progenitor cells (GPC) play key roles in the homeostatic renewal of gastric glands and are instrumental in epithelial repair after injury. Until very recently, the existence of GPC could only be inferred by indirect labeling strategies. The last few years have seen significant progress in the identification of biomarkers that allow prospective identification of GPC. The analysis of these unique cell populations is providing new insights into the molecular underpinnings of gastric epithelial homeostasis and repair. Of closely related interest is the potential to identify so-called cancer stem cells, a rare subpopulation of tumor-initiating cells. Here, we review the current useful biomarkers for GPC, including: (a) those that have been demonstrated by lineage tracing to give rise to all gastric cell lineages (e.g., the villin-transgene marker as well as Lgr5); (b) those that give rise to a subset of gastric lineages (e.g., TFF2); (c) markers that recognize cryptic progenitors for metaplasia (e.g., MIST1), and (d) markers that have not yet been analyzed by lineage tracing (e.g., DCKL1/DCAMKL1, CD133/PROM1, and CD44). The study of these markers has been mostly limited to the mouse model, but the hope is that the rapid pace of recent breakthroughs in this animal model will soon lead to a greater understanding of human gastric stem cell biology and to new insights into gastric cancer, the second leading cause of cancer-related death worldwide.
Asunto(s)
Células Madre Neoplásicas/metabolismo , Células Madre/metabolismo , Neoplasias Gástricas/metabolismo , Estómago/citología , Animales , Biomarcadores de Tumor/metabolismo , Humanos , Ratones , Factor Trefoil-2RESUMEN
Morphological development of the small intestinal mucosa involves the stepwise remodeling of a smooth-surfaced endodermal tube to form finger-like luminal projections (villi) and flask-shaped invaginations (crypts). These remodeling processes are orchestrated by instructive signals that pass bidirectionally between the epithelium and underlying mesenchyme. Sonic (Shh) and Indian (Ihh) hedgehog are expressed in the epithelium throughout these morphogenic events, and mice lacking either factor exhibit intestinal abnormalities. To examine the combined role of Shh and Ihh in intestinal morphogenesis, we generated transgenic mice expressing the pan-hedgehog inhibitor, Hhip (hedgehog interacting protein) in the epithelium. We demonstrate that hedgehog (Hh) signaling in the neonatal intestine is paracrine, from epithelium to Ptch1-expressing subepithelial myofibroblasts (ISEMFs) and smooth muscle cells (SMCs). Strong inhibition of this signal compromises epithelial remodeling and villus formation. Surprisingly, modest attenuation of Hh also perturbs villus patterning. Desmin-positive smooth muscle progenitors are expanded, and ISEMFs are mislocalized. This mesenchymal change secondarily affects the epithelium: Tcf4/beta-catenin target gene activity is enhanced, proliferation is increased, and ectopic precrypt structures form on villus tips. Thus, through a combined Hh signal to underlying ISEMFs, the epithelium patterns the crypt-villus axis, ensuring the proper size and location of the emerging precrypt compartment.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Mucosa Intestinal/metabolismo , Transactivadores/fisiología , Animales , Tipificación del Cuerpo , Proteína Morfogenética Ósea 4 , Proteínas Morfogenéticas Óseas/metabolismo , Diferenciación Celular , Proteínas del Citoesqueleto/metabolismo , ADN Complementario/metabolismo , Células Epiteliales/citología , Epitelio/metabolismo , Fibroblastos/metabolismo , Proteínas Hedgehog , Humanos , Inmunohistoquímica , Hibridación in Situ , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas de Microfilamentos/fisiología , Microscopía Fluorescente , Músculo Liso/citología , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Factores de Tiempo , Transactivadores/metabolismo , Transgenes , Proteínas Wnt , beta CateninaRESUMEN
Chronic Helicobacter pylori infection results in serious sequelae, including atrophy, intestinal metaplasia, and gastric cancer. Intestinal metaplasia in the stomach is defined by the presence of intestine-like cells expressing enterocyte-specific markers, such as villin. In this study, we demonstrate that villin is expressed in intestine-like cells that develop after chronic infection with H. pylori in both human stomach and in a mouse model. Transfection studies were used to identify specific regions of the villin promoter that are inducible by exposure of the cells to H. pylori. We demonstrated that induction of the villin promoter by H. pylori in a human gastric adenocarcinoma cell line (AGS) required activation of the Erk pathway. Elk-1 and the serum response factor (SRF) are downstream transcriptional targets of the Erk pathway. We observed inducible binding of Elk-1 and the SRF after 3 and 24 h of treatment with H. pylori, suggesting that the bacteria alone are sufficient to initiate a cascade of signaling events responsible for villin expression. Thus, H. pylori induction of villin in the stomach correlates with activation and cooperative binding of Elk-1 and the SRF to the proximal promoter of villin.
Asunto(s)
Proteínas de Unión al ADN/biosíntesis , Helicobacter pylori/metabolismo , Intestinos/microbiología , Metaplasia/microbiología , Proteínas de Microfilamentos/biosíntesis , Proteínas Proto-Oncogénicas/biosíntesis , Factor de Respuesta Sérica/metabolismo , Estómago/microbiología , Factores de Transcripción/biosíntesis , Animales , Secuencia de Bases , Línea Celular Tumoral , Técnicas de Cocultivo , Relación Dosis-Respuesta a Droga , Mucosa Gástrica/metabolismo , Genes Reporteros , Humanos , Mucosa Intestinal/metabolismo , Ratones , Ratones Endogámicos C57BL , Microscopía Fluorescente , Datos de Secuencia Molecular , Plásmidos/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Transducción de Señal , Factores de Tiempo , Transfección , Proteína Elk-1 con Dominio ets , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Villin, an actin bundling protein found in the apical brush border of absorptive tissues, is one of the first structural genes to be transcriptionally activated in the embryonic intestinal endoderm. In the adult, villin is broadly expressed in every cell of the intestinal epithelium on both the vertical axis (crypt to villus tip) and the horizontal axis (duodenum through colon) of the intestine. Here, we document that a 12.4-kilobase region of the mouse villin gene drives high level expression of two different reporter genes (LacZ and Cre recombinase) within the entire intestinal epithelium of transgenic mice. Deletion of a portion of this transgene results in reduction of beta-galactosidase activity in restricted domains of the small intestine (duodenum) and large intestine (cecum). In addition, expression is reduced in the crypt compartment throughout the intestine. Thus, the global expression pattern of villin in the intestine is apparently the consequence of an amalgam of distinct and individual domain-specific control processes. That is, expression of villin in the duodenum and cecum requires different regulatory sequences than the rest of the intestine, and the expression of villin in crypts is regulated by different circuitry than expression of villin on villus tips.
Asunto(s)
Proteínas Portadoras/genética , Ciego/metabolismo , Duodeno/metabolismo , Mucosa Intestinal/metabolismo , Proteínas de Microfilamentos/genética , Secuencias Reguladoras de Ácidos Nucleicos , Animales , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Endodermo/metabolismo , Eliminación de Gen , Regulación de la Expresión Génica , Mucosa Intestinal/anatomía & histología , Intestino Grueso/metabolismo , Intestino Delgado/metabolismo , Luciferasas/metabolismo , Ratones , Ratones Transgénicos , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/metabolismo , Modelos Genéticos , Estructura Terciaria de Proteína , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Transfección , beta-Galactosidasa/metabolismoRESUMEN
In the adult gastrointestinal tract, the morphologic borders between esophagus and stomach and between stomach and small intestine are literally one cell thick. The patterning mechanisms that underlie the development of these sharp regional divisions from a once continuous endodermal tube are still obscure. In the embryonic endoderm of the developing gut, region-specific expression of certain genes (e.g., intestine-specific expression of the actin bundling protein villin) can be detected as early as 9.0 days post coitum, although the morphologic differentiation of the gut epithelium proper does not begin until 4 to 5 days later. By using a mouse model in which a beta-galactosidase marker has been inserted into the endogenous villin locus, we examined the development of the stomach/intestinal (pyloric) border during gut organogenesis. The data indicate that the border is not sharp from the outset. Rather, the initial border region is characterized by a decreasing gradient of villin/beta-galactosidase expression that extends into the distal stomach. A sharp epithelial border of villin/beta-galactosidase expression appears abruptly at day 16 and is further refined over the next 3 weeks to form the distinct one-cell-thick border characteristic of the adult. These results indicate that an important previously unrecognized patterning event occurs in the gut epithelium at 16 days; this event may define an epithelial compartment boundary between the stomach and the intestine. The villin/beta-galactosidase mouse model characterized here provides an excellent substrate with which to further dissect the mechanisms involved in this patterning process.