RESUMEN
CD11b(+)Gr-1(+) myeloid-derived suppressor cells (MDSCs) expand in the spleen during cancer and promote progression through suppression of cytotoxic T cells. An anti-inflammatory reflex arc involving the vagus nerve and memory T cells is necessary for resolution of acute inflammation. Failure of this neural circuit could promote procarcinogenic inflammation and altered tumour immunity. Here we show that splenic TFF2, a secreted anti-inflammatory peptide, is released by vagally modulated memory T cells to suppress the expansion of MDSCs through CXCR4. Splenic denervation interrupts the anti-inflammatory neural arc, resulting in the expansion of MDSCs and colorectal cancer. Deletion of Tff2 recapitulates splenic denervation to promote carcinogenesis. Colorectal carcinogenesis could be suppressed through transgenic overexpression of TFF2, adenoviral transfer of TFF2 or transplantation of TFF2-expressing bone marrow. TFF2 is important to the anti-inflammatory reflex arc and plays an essential role in arresting MDSC proliferation. TFF2 offers a potential approach to prevent and to treat cancer.
Asunto(s)
Proliferación Celular/genética , Colitis/genética , Neoplasias Colorrectales/genética , Mucinas/genética , Proteínas Musculares/genética , Células Mieloides/inmunología , Péptidos/genética , Receptores CXCR4/inmunología , Bazo/inmunología , Linfocitos T Citotóxicos/inmunología , Nervio Vago , Traslado Adoptivo , Animales , Western Blotting , Trasplante de Médula Ósea , Colitis/inmunología , Colon/metabolismo , Colon/patología , Neoplasias Colorrectales/inmunología , Citocinas/inmunología , Desnervación , Progresión de la Enfermedad , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Inmunohistoquímica , Inflamación , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mucinas/inmunología , Mucinas/metabolismo , Proteínas Musculares/inmunología , Proteínas Musculares/metabolismo , Neoplasias/genética , Neoplasias/inmunología , Péptidos/inmunología , Péptidos/metabolismo , Permeabilidad , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Bazo/inervación , Subgrupos de Linfocitos T/inmunología , Factor Trefoil-2 , Vagotomía Troncal , Estimulación del Nervio VagoRESUMEN
Overexpression of trefoil factor 2 (TFF2) is associated with increased cell migration, resistance to apoptosis, and possibly increased gastric cancer invasion. Dysregulation of p53 is frequently observed in preneoplastic conditions of the stomach. Here, we investigated the effect of p53 on the expression and function of TFF2 in gastric cancer cell lines. Gene expression was determined by reverse transcription-polymerase chain reaction, and promoter activity was assessed by dual luciferase reporter assays. Apoptosis was detected by flow cytometry, and cell migration was evaluated by the Boyden chamber assay. Exogenous expression of p53 dose dependently inhibited endogenous TFF2 mRNA, protein, and promoter activity and resulted in induction of cell apoptosis and inhibition of cell migration. Downregulation of TFF2 by small interfering RNA sensitized gastric cancer cells to drug-induced p53-dependent apoptosis. Addition of human TFF2 peptide reversed p53-dependent apoptosis and inhibition of cell migration. The p53-responsive element was mapped to an AP-1-like cis-element at -182 bp upstream of the TFF2 transcription start site. Mutation of this AP-1-like element abrogated p53-mediated inhibition of TFF2 promoter activity. Gel shift and chromatin immunoprecipitation assays demonstrated that c-Jun and c-Fos bind to this AP-1-like element. Ectopic expression of c-Jun/c-Fos or p300 or treatment of cells with phorbol 12-myristate 13-acetate (PMA) stimulated endogenous TFF2 mRNA expression and promoter activity, and p53 inhibited the effects of AP-1 and PMA on TFF2. p53 induces cell apoptosis and inhibits cell migration in part by downregulating TFF2 expression through an AP-1-like site, suggesting that TFF2 may be an important downstream target of p53.
Asunto(s)
Apoptosis , Movimiento Celular , Mucinas/metabolismo , Proteínas Musculares/metabolismo , Péptidos/metabolismo , Neoplasias Gástricas/metabolismo , Factor de Transcripción AP-1/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Sitios de Unión , Línea Celular Tumoral , Regulación hacia Abajo , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Mucinas/genética , Proteínas Musculares/genética , Mutación , Péptidos/genética , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Interferencia de ARN , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Factor de Transcripción AP-1/genética , Transcripción Genética , Transfección , Factor Trefoil-2 , Proteína p53 Supresora de Tumor/genética , Factores de Transcripción p300-CBP/metabolismoRESUMEN
Trefoil family factor 2 (TFF2) is expressed in gastrointestinal epithelial cells where it serves to maintain mucosal integrity and promote epithelial repair. The peptide hormone, gastrin, stimulates acid secretion but also induces proliferation of the acid-secreting mucosa. Because the relationship between these peptides of overlapping function is not understood, we chose to investigate the regulatory effect of gastrin on TFF2 expression. The expression of mRNA and protein of TFF2 was determined by RT-PCR and immunohistochemical staining, respectively. A series of truncated and mutant murine TFF2 promoter constructs was generated. Promoter activity was assessed using dual luciferase reporter assays. Gastrin-responsive DNA-binding sites in the TFF2 promoter were evaluated by electrophoretic mobility shift assay. Gastrin significantly increased the level of endogenous mRNA of TFF2 in the gastrin receptor-expressing AGS-E gastric cancer cell line in a time- and dose-dependent manner. TFF2 protein expression in the gastric fundus was elevated in hypergastrinemic (INS-GAS) transgenic mice and reduced in gastrin-deficient mice. Gastrin treatment increased TFF2 promoter activity through cis-acting regions, containing CCAATA- and GC-rich enhancers. Pretreatment with Y-F476, a gastrin/CCK(B) receptor antagonist, abolished gastrin-dependent promoter activity. Inhibitors of protein kinase C (PKC), mitogen/extracellular signal-regulated kinase (MEK1), and phosphatidylinositol 3-kinase (PI 3-kinase) reduced gastrin-dependent TFF2 promoter activity, whereas an epithelial growth factor receptor (EGFR) inhibitor had no effect. We found that gastrin regulates TFF2 transcription through a GC-rich DNA-binding site and a PKC-, MEK1- and PI 3-kinase-dependent but EGFR-independent pathway. Regulation of TFF2 by gastrin may play a role in the maintenance and repair of the gastrointestinal mucosa.
