RESUMEN
In colorectal cancer, the antitumorigenic guanylyl cyclase C (GCC) signalome is defective reflecting ligand deprivation from downregulation of endogenous hormone expression. Although the proximal intracellular mediators of that signal transduction system, including cyclic guanosine monophosphate (cGMP) and cGMP-dependent protein kinase (PKG), are well characterized, the functional significance of its distal effectors remain vague. Dysregulation of ligand-dependent GCC signaling through vasodilator-stimulated phosphoprotein (VASP), an actin-binding protein implicated in membrane protrusion dynamics, drastically reduced cGMP-dependent VASP phosphorylation levels in colorectal tumors from patients. Restoration of cGMP-dependent VASP phosphorylation by GCC agonists suppressed the number and length of locomotory (filopodia) and invasive (invadopodia) actin-based organelles in human colon cancer cells. Membrane organelle disassembly reflected specific phosphorylation of VASP Ser239, the cGMP/PKG preferred site, and rapid VASP removal from tumor cell protrusions. Importantly, VASP Ser239 phosphorylation inhibited the proteolytic function of invadopodia, reflected by suppression of the cancer cell ability to digest DQ-collagen IV embedded in Matrigel. These results demonstrate a previously unrecognized role for VASP Ser239 phosphorylation, a single intracellular biochemical reaction, as an effective mechanism which opposes tumor cell shape promoting colon cancer invasion and metastasis. Reconstitution of physiological cGMP circuitry through VASP, in turn, represents an attractive targeted approach for patients with colorectal cancer.
Asunto(s)
Moléculas de Adhesión Celular/fisiología , Extensiones de la Superficie Celular/patología , Neoplasias del Colon/patología , Proteínas de Microfilamentos/fisiología , Fosfoproteínas/fisiología , Línea Celular Tumoral , Movimiento Celular , Forma de la Célula , Colágeno Tipo IV/metabolismo , Guanilato Ciclasa/fisiología , Humanos , Invasividad Neoplásica , Fosforilación , Transducción de SeñalRESUMEN
AIMS: Vasodilator-stimulated phosphoprotein (VASP) controls actin dynamics associated with the malignant phenotype of colorectal tumors. Oncogenic VASP function, in turn, is finely regulated by cyclic nucleotide-dependent phosphorylation of serine (Ser) residues 157 and 239, whose differential expression determines cell survival behavior in colon cancer. However, the role of differential VASP Ser phosphorylation in colorectal carcinogenesis remains unclear. MAIN METHODS: Specific VASP phosphomutant constructs were employed to selectively silence Ser157 or Ser239 phosphorylation in human colon carcinoma cells. Cyclic nucleotide-dependent manipulation of VASP Ser phosphorylation was performed with 8-bromoadenosine 3',5'-cyclic adenosine monophosphate (8-Br-cAMP) or 8-chlorophenylthio 3',5'-cyclic guanosine monophosphate (8-CPT-cGMP). Tumorigenic and locomotory phenotypes were examined in vitro with clonogenic and wound healing assays, respectively. Finally, tumor formation and growth were investigated in vivo employing two distinct xenograft models of colorectal cancer. KEY FINDINGS: Disruption of VASP Ser157 phosphorylation weakened the clonogenic and migratory abilities of human colon cancer cells, effects mimicked by 8-CPT-cGMP-dependent regulation of VASP Ser239. In contrast, inhibition of VASP Ser239 phosphorylation enhanced cell clonogenicity and migration and was phenocopied by 8-Br-cAMP-dependent regulation of VASP Ser157. Importantly, cancer cells bearing the phosphomutant construct targeting VASP Ser157 decreased, while those with the phosphomutation at Ser239 improved their abilities to establish productive tumor colonies and grow in the peritoneal cavity or subcutaneous tissues of nude mice. SIGNIFICANCE: Together, present observations suggest differential VASP Ser phosphorylation is a relevant, targetable molecular event underlying tumor formation and progression in colon cancer.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Moléculas de Adhesión Celular/metabolismo , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Proteínas de Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Serina/metabolismo , Animales , Movimiento Celular/fisiología , Proliferación Celular , Células HCT116 , Humanos , Masculino , Ratones , Ratones Desnudos , Fosforilación/fisiología , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
ABSTRACT: Novel foods, such as edible insects and food products on the basis of insects, could play an important role in both human and animal nutrition in the future. The identification of dangers associated with insect consumption is fundamental to guarantee consumer safety and adequate regulatory guidelines for operators of the food sector. Although former studies have focused on the microbiological contamination of fresh or processed edible insects, so far little information is available about the occurrence of foodborne parasites, such as Toxoplasma gondii, whose life cycles make them candidates for potential insect breeding substrate contamination. Hence, we investigated the presence of contaminating T. gondii in farmed edible insects to rule out this further hazard for consumers. Four species of insects most commonly used as food for human consumption were analyzed: mealworm; African migratory locust, house cricket, and silkworm. Samples included live specimens but also minimally (dehydrated) and highly processed edible insects. Traces of T. gondii DNA were detected in samples of dehydrated mealworm. These results highlight the need for implementing good farming and processing practices with particular care paid to safe storage and handling of feed and substrates used for edible insects to reduce the chance of T. gondii entering the human food chain.
Asunto(s)
Insectos Comestibles , Toxoplasma , Animales , Alimentos , Inocuidad de los Alimentos , Humanos , InsectosRESUMEN
Colorectal cancer is one of the leading causes of tumor-related morbidity and mortality worldwide. While mechanisms underlying this disease have been elucidated over the past two decades, these molecular insights have failed to translate into efficacious therapy. The oncogenomic view of cancer suggests that terminal transformation reflects the sequential corruption of signal transduction circuits regulating key homeostatic mechanisms, whose multiplicity underlies the therapeutic resistance of most tumors to interventions targeting individual pathways. Conversely, the paucity of mechanistic insights into proximal pathophysiological processes that initiate and amplify oncogenic circuits preceding accumulation of mutations and transformation impedes development of effective prevention and therapy. In that context, guanylyl cyclase C (GCC), the intestinal receptor for the paracrine hormones guanylin and uroguanylin, whose early loss characterizes colorectal transformation, has emerged as a component of lineage-specific homeostatic programs organizing spatiotemporal patterning along the crypt-surface axis. Dysregulation of GCC signaling, reflecting hormone loss, promotes tumorigenesis through reprogramming of replicative and bioenergetic circuits and genomic instability. Compensatory upregulation of GCC in response to hormone loss provides a unique translational opportunity for prevention and treatment of colorectal tumors by hormone-replacement therapy.
Asunto(s)
Transformación Celular Neoplásica/genética , Neoplasias Colorrectales/enzimología , Neoplasias Colorrectales/genética , Predisposición Genética a la Enfermedad , Guanilato Ciclasa/genética , Receptores de Péptidos/genética , Animales , Humanos , Receptores de Enterotoxina , Receptores Acoplados a la Guanilato-CiclasaRESUMEN
Guanylyl cyclase C (GCC), the receptor for diarrheagenic bacterial heat-stable enterotoxins (STs), inhibits colorectal cancer cell proliferation by co-opting Ca(2+) as the intracellular messenger. Similarly, extracellular Ca(2+) (Ca(2+)(o)) opposes proliferation and induces terminal differentiation in intestinal epithelial cells. In that context, human colon cancer cells develop a phenotype characterized by insensitivity to cytostasis imposed by Ca(2+)(o). Here, preconditioning with ST, mediated by GCC signaling through cyclic nucleotide-gated channels, restored Ca(2+)(o)-dependent cytostasis, reflecting posttranscriptional regulation of calcium-sensing receptors (CaRs). ST-induced GCC signaling deployed CaRs to the surface of human colon cancer cells, whereas elimination of GCC signaling in mice nearly abolished CaR expression in enterocytes. Moreover, ST-induced Ca(2+)(o)-dependent cytostasis was abrogated by CaR-specific antisense oligonucleotides. Importantly, following ST preconditioning, newly expressed CaRs at the cell surface represented tumor cell receptor targets for antiproliferative signaling by CaR agonists. Since expression of the endogenous paracrine hormones for GCC is uniformly lost early in carcinogenesis, these observations offer a mechanistic explanation for the Ca(2+)(o)-resistant phenotype of colon cancer cells. Restoration of antitumorigenic CaR signaling by GCC ligand replacement therapy represents a previously unrecognized paradigm for the prevention and treatment of human colorectal cancer employing dietary Ca(2+) supplementation.
Asunto(s)
División Celular/efectos de los fármacos , Neoplasias del Colon/patología , Enterotoxinas/farmacología , Receptores Sensibles al Calcio/fisiología , Apoptosis , Línea Celular Tumoral , Transformación Celular Neoplásica , Neoplasias Colorrectales/patología , Humanos , Precondicionamiento Isquémico , Necrosis , Receptores Sensibles al Calcio/efectos de los fármacosRESUMEN
BACKGROUND AND AIMS: The benefit of adjuvant chemotherapy for stage II colorectal cancer (CRC) patients remains unclear, emphasizing the need for improved prognostic biomarkers to identify patients at risk of metastatic recurrence. To address this unmet clinical need, we examined the expression and phosphorylation status of the vasodilator-stimulated phosphoprotein (VASP) in CRC tumor progression. VASP, a processive actin polymerase, promotes the formation of invasive membrane structures leading to extracellular matrix remodeling and tumor invasion. Phosphorylation of VASP serine (Ser) residues 157 and 239 regulate VASP function, directing subcellular localization and inhibiting actin polymerization, respectively. METHODS: The expression levels of VASP protein, pSer157-VASP, and pSer239-VASP were determined by immunohistochemistry in tumors and matched normal adjacent tissue from 141 CRC patients, divided into 2 cohorts, and the association of VASP biomarker expression with clinicopathologic features and disease recurrence was examined. RESULTS: We report that changes in VASP expression and phosphorylation were significantly associated with tumor invasion and disease recurrence. Furthermore, we disclose a novel 2-tiered methodology to maximize VASP positive and negative predictive value performance for prognostication. CONCLUSION: VASP biomarkers may serve as prognostic biomarkers in CRC and should be evaluated in a larger clinical study.
RESUMEN
BACKGROUND: The current paradigm suggests that matrix metalloproteinase 9 (MMP-9) expressed by stromal cells is a therapeutic target in human colorectal tumors which presumably regulates metastatic disease progression. Conversely, whereas cancer cells within those tumors may induce stromal cells to produce MMP-9 and may be targets for MMP-9 activity, they are not the source of MMP-9 underlying metastasis. METHODS: MMP-9 expression in matched colorectal tumors and normal adjacent mucosa from patients and human colon cancer cell lines was examined by real-time reverse transcription-PCR, laser capture microdissection, immunoelectron microscopy, and immunoblot analysis. The role of colon cancer cell MMP-9 in processes underlying metastasis was explored in vitro by examining degradation of extracellular matrix components by gelatin zymography and formation of locomotory organelles by cell spreading analysis and in vivo by quantifying hematogenous tumor cell seeding of mouse lungs. RESULTS: Primary colorectal tumors overexpress MMP-9 compared with matched normal adjacent mucosa. In contrast to the current paradigm, MMP-9 is expressed equally by cancer and stromal cells within human colon tumors. Cancer cell MMP-9 regulates metastatic behavior in vitro, including degradation of extracellular matrix components and formation of locomotory organelles. Moreover, this MMP-9 critically regulates hematogenous seeding of mouse lungs by human colon cancer cells in vivo. CONCLUSIONS: These observations reveal that MMP-9 produced by human colon cancer, rather than stromal, cells is central to processes underlying metastasis. They underscore the previously unrecognized potential of specifically targeting tumor cell MMP-9 in interventional strategies to reduce mortality from metastatic colorectal cancer.
Asunto(s)
Antineoplásicos/uso terapéutico , Neoplasias Colorrectales/tratamiento farmacológico , Metaloproteinasa 9 de la Matriz/genética , Metástasis de la Neoplasia/prevención & control , Adulto , Anciano , Anciano de 80 o más Años , Animales , Antineoplásicos/farmacología , Células CACO-2 , Línea Celular Tumoral , Neoplasias Colorrectales/enzimología , Neoplasias Colorrectales/patología , Células Epiteliales/enzimología , Células Epiteliales/patología , Células Epiteliales/ultraestructura , Femenino , Expresión Génica/genética , Humanos , Immunoblotting , Masculino , Metaloproteinasa 9 de la Matriz/metabolismo , Inhibidores de la Metaloproteinasa de la Matriz , Ratones , Ratones Desnudos , Microscopía Inmunoelectrónica , Persona de Mediana Edad , Metástasis de la Neoplasia/patología , Neoplasias Experimentales/enzimología , Neoplasias Experimentales/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Bacterial diarrheagenic heat-stable enterotoxins induce colon cancer cell cytostasis by targeting guanylyl cyclase C (GCC) signaling. Anticancer actions of these toxins are mediated by cyclic guanosine 3',5'-monophosphate (cGMP)-dependent influx of Ca2+ through cyclic nucleotide-gated channels. However, prolonged stimulation of GCC produces resistance in tumor cells to heat-stable enterotoxin-induced cytostasis. Resistance reflects rapid (tachyphylaxis) and slow (bradyphylaxis) mechanisms of desensitization induced by cGMP. Tachyphylaxis is mediated by cGMP-dependent protein kinase, which limits the conductance of cyclic nucleotide-gated channels, reducing the influx of Ca2+ propagating the antiproliferative signal from the membrane to the nucleus. In contrast, bradyphylaxis is mediated by cGMP-dependent allosteric activation of phosphodiesterase 5, which shapes the amplitude and duration of heat-stable enterotoxin-dependent cyclic nucleotide accumulation required for cytostasis. Importantly, interruption of tachyphylaxis and bradyphylaxis restores cancer cell cytostasis induced by heat-stable enterotoxins. Thus, regimens that incorporate cytostatic bacterial enterotoxins and inhibitors of cGMP-mediated desensitization offer a previously unrecognized therapeutic paradigm for treatment and prevention of colorectal cancer.
Asunto(s)
Toxinas Bacterianas/farmacología , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/metabolismo , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Enterotoxinas/farmacología , Hidrolasas Diéster Fosfóricas/metabolismo , 3',5'-GMP Cíclico Fosfodiesterasas , Calcio/metabolismo , Línea Celular Tumoral , Neoplasias del Colon/enzimología , Neoplasias del Colon/patología , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacología , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5 , Proteínas de Escherichia coli , Guanilato Ciclasa/metabolismo , Humanos , Receptores de Enterotoxina , Receptores Acoplados a la Guanilato-Ciclasa , Receptores de Péptidos/metabolismo , Transducción de SeñalRESUMEN
AIMS: In colon cancer, disease recurrence and death are associated with abnormal tumor cell survival. Vasodilator-stimulated phosphoprotein (VASP) is an actin binding protein regulating cell shape and polarity through the F-actin cytoskeleton, whose activity is controlled by cAMP-dependent phosphorylation at Ser157 and cGMP-dependent phosphorylation at Ser239. This study examined the role of differential VASP Ser phosphorylation in regulating cell survival and apoptosis in human colon carcinoma cells. MAIN METHODS: Selective inhibition of VASP Ser157 or Ser239 phosphorylation in colon cancer cells was performed with specific phosphomutant constructs. F-actin organization was examined by confocal microscopy, and the balance of cell survival and death assessed by measuring acridine orange and ethidium bromide staining, caspase-3 and BAD-pS112 expression and DNA fragmentation. KEY FINDINGS: In human colon carcinoma cells suppression of VASP Ser157 phosphorylation reduced F-actin content and survival and increased apoptosis, while inhibition of VASP Ser239 phosphorylation increased F-actin content and survival and reduced cell death. Also, while 8Br-cAMP induced VASP Ser157 phosphorylation and reduced cell death, treatments with 8CPT-cGMP elevated VASP Ser239 phosphorylation and promoted apoptosis. SIGNIFICANCE: These findings suggest that differential VASP Ser phosphorylation represents a unique therapeutic target to control cell survival and death behavior in colon cancer. In particular, pharmacological manipulation of VASP Ser phosphorylation could be exploited to affect the malignant actin cytoskeleton and induce apoptosis in colorectal cancer cells.
Asunto(s)
Apoptosis/fisiología , Moléculas de Adhesión Celular/metabolismo , Supervivencia Celular/fisiología , Neoplasias del Colon/metabolismo , Proteínas de Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Serina/metabolismo , Naranja de Acridina , Actinas/química , Western Blotting , Caspasa 3/metabolismo , Línea Celular Tumoral , Fragmentación del ADN , Etidio , Humanos , Microscopía Confocal , FosforilaciónRESUMEN
Inhibition of ex vivo arachidonic acid (AA)-induced aggregation is a biomarker for the isotype selectivity of cyclooxygenase (COX) inhibitors since platelets express COX-1 but not COX-2. At low concentrations, there is broad inter- and intrasubject variability in AA-induced aggregation of platelets ex vivo. This study defined a concentration that reliably induces aggregation without overcoming inhibition by therapeutic aspirin therapy (ASA, 81-mg) treatment. Logistic regression analysis of ex vivo aggregation, induced with increasing concentrations of AA in platelet-rich plasma (PRP), estimated that platelets from > or = 90% of subjects would aggregate at > or = 1.5 mM AA (95% confidence interval [CI], 1.1, 2.1). A concentration of 1.6 mM AA failed to aggregate platelets from 26 healthy volunteers, who had previously aggregated at this concentration, following six daily oral doses of 81 mg of ASA. These data demonstrate that 1.6 mM AA reproducibly induces platelet aggregation in PRP from healthy volunteers without overcoming the antiplatelet effect of daily low-dose aspirin therapy.
Asunto(s)
Ácido Araquidónico/farmacología , Agregación Plaquetaria/efectos de los fármacos , Prostaglandina-Endoperóxido Sintasas/sangre , Adolescente , Adulto , Ácido Araquidónico/sangre , Aspirina/administración & dosificación , Aspirina/farmacología , Inhibidores de la Ciclooxigenasa/administración & dosificación , Inhibidores de la Ciclooxigenasa/farmacología , Esquema de Medicación , Interacciones Farmacológicas , Femenino , Humanos , Masculino , Persona de Mediana Edad , Reproducibilidad de los ResultadosRESUMEN
Agonists of the transmembrane intestinal receptor guanylyl cyclase C (GCC) have recently attracted interest as promising human therapeutics. Peptide ligands that can specifically induce GCC signaling in the intestine include endogenous hormones guanylin and uroguanylin, diarrheagenic bacterial enterotoxins (ST), and synthetic drugs linaclotide, plecanatide, and SP-333. These agonists bind to GCC at intestinal epithelial surfaces and activate the receptor's intracellular catalytic domain, an event initiating discrete biological responses upon conversion of guanosine-5'-triphosphate to cyclic guanosine monophosphate. A principal action of GCC agonists in the colon is the promotion of mucosal homeostasis and its dependent barrier function. Herein, GCC agonists are being developed as new medications to treat inflammatory bowel diseases, pathological conditions characterized by mucosal barrier hyperpermeability, abnormal immune reactions, and chronic local inflammation. This review will present important concepts underlying the pharmacology and therapeutic utility of GCC agonists for patients with ulcerative colitis, one of the most prevalent inflammatory bowel disease disorders.
Asunto(s)
Colitis Ulcerosa/tratamiento farmacológico , Receptores Acoplados a la Guanilato-Ciclasa/agonistas , Receptores de Péptidos/agonistas , Toxinas Bacterianas/farmacología , Enterotoxinas/farmacología , Proteínas de Escherichia coli/farmacología , Humanos , Péptidos/uso terapéutico , Receptores de Enterotoxina , Receptores Acoplados a la Guanilato-Ciclasa/fisiología , Receptores de Péptidos/fisiología , Transducción de Señal/fisiologíaRESUMEN
Matrix metalloproteinase-9 (MMP-9) produced by colorectal cancer cells is a critical determinant of metastatic disease progression and an attractive target for antimetastatic strategies to reduce colon cancer mortality. Cellular signaling by cyclic GMP (cGMP) regulates MMP-9 dynamics in various cell systems, and the bacterial enterotoxin receptor guanylyl cyclase C (GCC), the principle source of cGMP in colonocytes, which is overexpressed in colorectal cancers, inhibits tumor initiation and progression in the intestine. Here, we show that ligand-dependent GCC signaling through cGMP induces functional remodeling of cancer cell MMP-9 reflected by a compartmental redistribution of this gelatinase, in which intracellular retention resulted in reciprocal extracellular depletion. Functional remodeling of MMP-9 by GCC signaling reduced the ability of colon cancer cells to degrade matrix components, organize the actin cytoskeleton to form locomotory organelles and spread, and hematogenously seed distant organs. Of significance, GCC effects on cancer cell MMP-9 prevented establishment of metastatic colonies by colorectal cancer cells in the mouse peritoneum in vivo. Because endogenous hormones for GCC are uniformly deficient in intestinal tumors, reactivation of dormant GCC signaling with exogenous administration of GCC agonists may represent a specific intervention to target MMP-9 functions in colon cancer cells. The notion that GCC-mediated regulation of cancer cell MMP-9 disrupts metastasis, in turn, underscores the unexplored utility of GCC hormone replacement therapy in the chemoprevention of colorectal cancer progression.
Asunto(s)
Neoplasias del Colon/enzimología , Neoplasias del Colon/patología , Guanilato Ciclasa/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Receptores de Péptidos/metabolismo , Células CACO-2 , Línea Celular Tumoral , Movimiento Celular/fisiología , GMP Cíclico/metabolismo , Células Epiteliales/enzimología , Humanos , Inhibidores de la Metaloproteinasa de la Matriz , Metástasis de la Neoplasia , Neoplasias Peritoneales/enzimología , Neoplasias Peritoneales/secundario , Receptores de Enterotoxina , Receptores Acoplados a la Guanilato-Ciclasa , Transducción de SeñalRESUMEN
BACKGROUND AND AIMS: Ovarian hormones oppose colorectal cancer, although mechanisms remain undefined. Similarly, the most commonly lost gene products in intestinal neoplasia include guanylin and uroguanylin, paracrine hormones for guanylyl cyclase C (GCC), which recently emerged as a tumor suppressor. However, the molecular intersection between intestinal paracrine and systemic sex hormones opposing intestinal neoplasia has not been explored. METHODS: Intestinal tumorigenesis was quantified in wild type (Gcc(+/+)) and GCC-deficient (Gcc(-/-)) mice carrying mutations in adenomatous polyposis coli (Apc) (Apc(Min/+)) or exposed to the carcinogen azoxymethane (AOM). Proliferation of epithelial cells was examined employing cell cycle markers. RESULTS: Deletion of Gcc increased tumor multiplicity and growth in colons and small intestines, respectively, of Apc(Min) (/+) mice. While changes in multiplicity and growth increased tumor burden, females exhibited approximately 60% (p= 0.040) of the burden in males. Similarly, female Gcc(-/-) mice treated with AOM exhibited approximately 40% (p= 0.048) of the burden in males. Moreover, Gcc deletion promoted epithelial cell proliferation, quantified by increases in beta-catenin, cMyc, cyclin D1, and phosphorylated retinoblastoma protein (pRb), in males but not females. CONCLUSION: There is a previously unappreciated interaction between sex and GCC signaling restricting crypt cell proliferation. Thus, the invariable loss of guanylin and uroguanylin resulting in tumorigenesis is mitigated in females by hormonal components of the ovarian axis. In the context of the universal overexpression of GCC by tumors, these observations highlight the combination of GCC paracrine and ovarian hormones for targeted prevention and therapy of colorectal cancer.
Asunto(s)
Transformación Celular Neoplásica/patología , Guanilato Ciclasa/metabolismo , Intestinos/enzimología , Intestinos/patología , Receptores de Péptidos/metabolismo , Caracteres Sexuales , Proteínas Supresoras de Tumor/metabolismo , Proteína de la Poliposis Adenomatosa del Colon/metabolismo , Animales , Azoximetano , Proliferación Celular , Neoplasias Colorrectales/enzimología , Neoplasias Colorrectales/patología , Enterocitos/enzimología , Enterocitos/patología , Femenino , Masculino , Ratones , Receptores de Enterotoxina , Receptores Acoplados a la Guanilato-CiclasaRESUMEN
Guanylyl cyclase C (GC-C), the receptor for diarrheagenic enterotoxins and the paracrine ligands guanylin and uroguanylin, regulates intestinal secretion. Beyond volume homeostasis, its importance in modulating cancer cell proliferation and its uniform dysregulation early in colon carcinogenesis, reflecting loss of ligand expression, suggests a role for GC-C in organizing the crypt-villus axis. Here, eliminating GC-C expression in mice increased crypt length along a decreasing rostral-caudal gradient by disrupting component homeostatic processes. Crypt expansion reflected hyperplasia of the proliferating compartment with reciprocal increases in rapidly cycling progenitor cells and reductions in differentiated cells of the secretory lineage, including Paneth and goblet cells, but not enteroendocrine cells. GC-C signaling regulated proliferation by restricting the cell cycle at the G(1)/S transition. Moreover, crypt expansion in GC-C(-/-) mice was associated with adaptive increases in cell migration and apoptosis. Reciprocal alterations in proliferation and differentiation resulting in expansion associated with adaptive responses in migration and apoptosis suggest that GC-C coordinates component processes maintaining homeostasis of the crypt progenitor compartment. In the context of uniform loss of GC-C signaling during tumorigenesis, dysregulation of those homeostatic processes may contribute to mechanisms underlying colon cancer.
Asunto(s)
Proliferación Celular , Colon/patología , Guanilato Ciclasa/fisiología , Receptores de Péptidos/fisiología , Animales , Apoptosis/genética , Ciclo Celular/genética , Diferenciación Celular/genética , Movimiento Celular/genética , Guanilato Ciclasa/genética , Homeostasis/genética , Hiperplasia , Intestinos/patología , Ratones , Receptores de Enterotoxina , Receptores Acoplados a la Guanilato-Ciclasa , Receptores de Péptidos/genéticaRESUMEN
BACKGROUND AND AIMS: The most commonly lost gene products in colorectal carcinogenesis include guanylin and uroguanylin, endogenous ligands for guanylyl cyclase C (GCC). Beyond intestinal fluid balance, GCC mediates diarrhea induced by bacterial enterotoxins, and an inverse relationship exists between enterotoxigenic Escherichia coli infections producing the exogenous GCC ligand ST and colorectal cancer worldwide. However, the role of GCC in neoplasia remains obscure. METHODS: Intestinal tumorigenesis was examined in wild-type (Gcc(+/+)) and GCC-deficient (Gcc(-/-)) mice carrying mutations in Apc (Apc(Min/+)) or exposed to the carcinogen azoxymethane. Markers of DNA damage, loss of Apc heterozygosity, and beta-catenin mutations were used to assess genomic integrity. Hyperproliferation was explored using Ki67 and cell cycle markers. Apoptosis was quantified by transferase biotin-dUTP nick end labeling analysis. RESULTS: In colons of Apc(Min/+) mice, deletion of Gcc increased tumor incidence and multiplicity, reflecting uncoupling of loss of genomic integrity and compensatory apoptosis. Conversely, in the small intestine, elimination of Gcc increased tumorigenesis by enhancing proliferation without altering genomic integrity. Moreover, these distinct but mutually reinforcing mechanisms collaborate in azoxymethane-exposed mice, and deletion of Gcc increased tumor initiation and growth associated with hypermutation and hyperproliferation, respectively, in conjunction with attenuated apoptosis. CONCLUSIONS: GCC suppresses tumor initiation and growth by maintaining genomic integrity and restricting proliferation. This previously unrecognized role of GCC in inhibiting tumorigenesis, together with the invariant disruption in guanylin and uroguanylin expression early in carcinogenesis, and the uniform over-expression of GCC by tumors, underscores the potential of oral administration of GCC ligands for targeted prevention and therapy of colorectal cancer.
Asunto(s)
Proliferación Celular , Transformación Celular Neoplásica/metabolismo , Neoplasias del Colon/enzimología , Regulación Neoplásica de la Expresión Génica , Genes APC , Guanilato Ciclasa/metabolismo , Neoplasias Intestinales/enzimología , Intestino Delgado/enzimología , Receptores de Péptidos/metabolismo , Animales , Apoptosis , Azoximetano , Proteínas de Ciclo Celular/análisis , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Neoplasias del Colon/inducido químicamente , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Daño del ADN , Modelos Animales de Enfermedad , Guanilato Ciclasa/deficiencia , Guanilato Ciclasa/genética , Neoplasias Intestinales/inducido químicamente , Neoplasias Intestinales/genética , Neoplasias Intestinales/patología , Intestino Delgado/patología , Antígeno Ki-67/análisis , Pérdida de Heterocigocidad , Ratones , Ratones Noqueados , Mutación , Receptores de Enterotoxina , Receptores Acoplados a la Guanilato-Ciclasa , Receptores de Péptidos/deficiencia , Receptores de Péptidos/genética , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
BACKGROUND & AIMS: Although progression to adenocarcinoma at the gastroesophageal junction reflects exposure to acid and bile acids associated with reflux, mechanisms mediating this transformation remain undefined. Guanylyl cyclase C (GC-C), an intestine-specific tumor suppressor, may represent a mechanism-based marker and target of transformation at the gastroesophageal junction. The present studies examine the expression of GC-C in normal tissues and tumors from esophagus and stomach and mechanisms regulating its expression by acid and bile acids. METHODS: Gene expression was examined by reverse-transcription polymerase chain reaction, promoter analysis, immunohistochemistry, immunoblotting, and functional analysis. Promoter transactivation was quantified by using luciferase constructs and mutational analysis. DNA binding of transcription factors was examined by electromobility shift analysis. RESULTS: GC-C mRNA and protein were ectopically expressed in approximately 80% of adenocarcinomas arising in, but not in normal, esophagus and stomach. Similarly, in OE19 human esophageal cancer cells, deoxycholate and acid induced expression of GC-C. This was associated with the induction of expression of Cdx2, a transcription factor required for GC-C expression. In turn, induction of Cdx2 expression by deoxycholate was mediated by binding sites in the proximal promoter for nuclear factor kappaB (NF-kappaB). Furthermore, deoxycholate increased NF-kappaB activity, associated with nuclear translocation and Cdx2 promoter binding of the NF-kappaB subunit p50. Moreover, a dominant negative construct for NF-kappaB prevented deoxycholate-induced p50 nuclear translocation and activation of the Cdx2 promoter. CONCLUSIONS: Transformation associated with reflux at the gastroesophageal junction reflects activation by bile acid and acid of a transcriptional program involving NF-kappaB and Cdx2, which mediate intestinal metaplasia and ectopic expression of GC-C.
Asunto(s)
Ácidos y Sales Biliares/farmacología , Transformación Celular Neoplásica , Esófago/metabolismo , Guanilato Ciclasa/metabolismo , Proteínas de Homeodominio/metabolismo , Intestinos/enzimología , FN-kappa B/metabolismo , Receptores de Péptidos/metabolismo , Adenocarcinoma/enzimología , Factor de Transcripción CDX2 , Línea Celular Tumoral , Ácido Desoxicólico/farmacología , Neoplasias Esofágicas/enzimología , Unión Esofagogástrica/enzimología , Unión Esofagogástrica/metabolismo , Esófago/enzimología , Esófago/patología , Expresión Génica , Guanilato Ciclasa/genética , Proteínas de Homeodominio/genética , Humanos , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Receptores de Enterotoxina , Receptores Acoplados a la Guanilato-Ciclasa , Receptores de Péptidos/genética , Distribución Tisular/efectos de los fármacos , Transcripción Genética/efectos de los fármacosRESUMEN
Staging patients with colorectal cancer defines their prognosis and therapeutic management. Unfortunately, histopathology, the current standard for staging, is relatively insensitive for detecting occult micrometastases and a significant fraction of patients are understaged and, consequently, undertreated. Similarly, current approaches to postoperative surveillance of patients with colorectal cancer detect disease recurrence at a point when interventions have little impact on survival. The detection of rare cells in tissue, for accurately staging patients, and in blood, for detecting disease recurrence, could be facilitated by employing sensitive and specific markers of disease. Guanylyl cyclase C (GCC), the receptor for the diarrheagenic bacterial heat-stable enterotoxin, is expressed selectively by cells derived from intestinal mucosa, including normal intestinal cells and colorectal tumor cells, but not by extragastrointestinal tissues and tumors. The nearly uniform expression of relatively high levels by metastatic colorectal tumors suggests that GCC may be a sensitive and specific molecular marker for metastatic colorectal cancer cells. Employing GCC reverse transcriptase PCR, occult colorectal cancer micrometastases were detected in lymph nodes that escaped detection by histopathology. Moreover, marker expression correlated with the risk of disease recurrence. Similarly, GCC reverse transcriptase PCR revealed the presence of tumor cells in blood of all patients examined with metastatic colorectal cancer and, in some studies, was associated with an increased risk of disease recurrence and mortality. These observations suggest that GCC reverse transcriptase PCR is a sensitive and specific technique for identifying tumor cells in extraintestinal sites and may be useful for staging and postoperative surveillance of patients with colorectal cancer.
Asunto(s)
Biomarcadores de Tumor/análisis , Neoplasias Colorrectales/enzimología , Neoplasias Colorrectales/patología , Guanilato Ciclasa/metabolismo , Receptores de Péptidos/metabolismo , Animales , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/metabolismo , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/cirugía , Regulación Neoplásica de la Expresión Génica , Guanilato Ciclasa/análisis , Guanilato Ciclasa/sangre , Humanos , Metástasis Linfática/patología , Estadificación de Neoplasias , Receptores de Enterotoxina , Receptores Acoplados a la Guanilato-Ciclasa , Receptores de Péptidos/análisis , Receptores de Péptidos/sangreRESUMEN
Guanylyl cyclase C and accumulation of cGMP induced by bacterial heat-stable enterotoxins (STs) promote colon cancer cell cytostasis, serving as a tumor suppressor in intestine. Conversely, capacitative calcium entry through store-operated calcium channels (SOCs) is a key signaling mechanism that promotes colon cancer cell proliferation. The present study revealed that proliferative signaling by capacitative calcium entry through SOCs opposes and is reciprocally coupled to cytostasis mediated by guanylyl cyclase C in T84 human colon carcinoma cells. Elimination of capacitative calcium entry employing 2-aminoethoxydiphenylborate (2-APB), a selective inhibitor of SOCs, potentiated cytostasis induced by ST. Opposition of ST-induced cytostasis by capacitative calcium entry reflects reciprocal inhibition of guanylyl cyclase C signaling. Calcium entry through SOCs induced by the calcium-ATPase inhibitor thapsigargin or the receptor agonists UTP or carbachol inhibited guanylyl cyclase C-dependent cGMP accumulation. This effect was mimicked by the calcium ionophore ionomycin and blocked by 2-APB and intracellular 1,2-bis(o-amino-5,5'-dibromophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM), a chelator of calcium. Moreover, regulation by capacitative calcium entry reflected ligand-dependent sensitization of guanylyl cyclase C to inhibition by that cation. Although basal catalytic activity was refractory, ST-stimulated guanylyl cyclase C was inhibited by calcium, which antagonized binding of magnesium to allosteric sites required for receptor-effector coupling. These observations demonstrate that reciprocal regulation of guanylyl cyclase C signaling by capacitative calcium entry through SOCs represents one limb of a coordinated mechanism balancing colon cancer cell proliferation and cytostasis. They suggest that combining guanylyl cyclase C agonists and SOC inhibitors offers a novel paradigm for cGMP-directed therapy and prevention for colorectal tumors.
Asunto(s)
Toxinas Bacterianas/farmacología , Canales de Calcio/fisiología , Neoplasias del Colon/patología , Enterotoxinas/farmacología , Calcio/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neoplasias del Colon/tratamiento farmacológico , GMP Cíclico/fisiología , Proteínas de Escherichia coli , Guanilato Ciclasa/fisiología , Humanos , Transducción de SeñalRESUMEN
Calcium and guanosine-3',5'-cyclic monophosphate (cGMP) are second messenger molecules that regulate opposing physiological functions, reflected in the reciprocal regulation of their intracellular concentrations, in many systems. Indeed, cGMP and Ca2+ constitute discrete points of integration between multiple cell signaling cascades in both convergent and parallel pathways. This chapter describes the molecular mechanisms regulating intracellular Ca2+ and cGMP, and their integration in specific cellular responses.
Asunto(s)
Señalización del Calcio/fisiología , Calcio/fisiología , GMP Cíclico/fisiología , Animales , HumanosRESUMEN
Nitric oxide (NO), the principal endogenous ligand for soluble guanylate cyclase (sGC), stimulates that enzyme and accumulation of intracellular cGMP, which mediates many of the (patho) physiological effects of NO. Previous studies demonstrated that 2-substituted adenine nucleotides, including 2-methylthioATP (2MeSATP) and 2-chloroATP (2ClATP), allosterically inhibit guanylate cyclase C, the membrane-bound receptor for the Escherichia coli heat-stable enterotoxin in the intestine. The present study examined the effects of 2-substituted adenine nucleotides on crude and purified sGC. 2-Substituted nucleotides inhibited basal and NO-activated crude and purified sGC, when Mg2+ served as the substrate cation cofactor. Similarly, 2-substituted adenine nucleotides inhibited those enzymes when Mn2+, which activates sGC in a ligand-independent fashion, served as the substrate cation cofactor. Inhibition of sGC by 2-substituted nucleotides was associated with a decrease in Vmax, consistent with a noncompetitive mechanism. In contrast to guanylate cyclase C, 2-substituted nucleotides inhibited sGC by a guanine nucleotide-independent mechanism. These studies demonstrate that 2-substituted adenine nucleotides allosterically inhibit basal and ligand-stimulated sGC. They support the suggestion that allosteric inhibition by adenine nucleotides is a general characteristic of the family of guanylate cyclases. This allosteric inhibition is mediated by direct interaction of adenine nucleotides with sGC, likely at the catalytic domain in a region outside the substrate-binding site.