Guanylyl Cyclase C Research Articles

Cure and Curse: E. coli Heat-Stable Enterotoxin and Its Receptor Guanylyl Cyclase C

Enterotoxigenic Escherichia coli (ETEC) associated diarrhea is responsible for roughly half a million deaths per year, the majority taking place in developing countries. The main agent responsible for these diseases is the bacterial heat-stable enterotoxin STa. STa is secreted by ETEC and after secretion binds to the intestinal receptor guanylyl cyclase C (GC-C), thus triggering a signaling cascade that eventually leads to the release of electrolytes and water in the intestine. Additionally, GC-C is a specific marker for colorectal carcinoma and STa is suggested to have an inhibitory effect on intestinal carcinogenesis. To understand the conformational events involved in ligand binding to GC-C and to devise therapeutic strategies to treat both diarrheal diseases and colorectal cancer, it is paramount to obtain structural information on the receptor ligand system. Here we summarize the currently available structural data and report on physiological consequences of STa binding to GC-C in intestinal epithelia and colorectal carcinoma cells.

Bacterial Heat-Stable Enterotoxins: Translation of Pathogenic Peptides into Novel Targeted Diagnostics and Therapeutics

Heat-stable toxins (STs) produced by enterotoxigenic bacteria cause endemic and traveler’s diarrhea by binding to and activating the intestinal receptor guanylyl cyclase C (GC-C). Advances in understanding the biology of GC-C have extended ST from a diarrheagenic peptide to a novel therapeutic agent. Here, we summarize the physiological and pathophysiological role of GC-C in fluid-electrolyte regulation and intestinal crypt-villus homeostasis, as well as describe translational opportunities offered by STs, reflecting the unique characteristics of GC-C, in treating irritable bowel syndrome and chronic constipation, and in preventing and treating colorectal cancer.

Lack of Guanylate Cyclase C results in increased mortality in mice following liver injury

BackgroundGuanylate Cyclase C (GC-C) expression in the intestine plays a role in the regulation of fluid and ion transport, as well as epithelial cell apoptosis and proliferation. In the adult rat liver, GC-C expression is increased in response to injury. We hypothesized that GC-C is required for repair/recovery from liver injury.MethodsWe subjected wild type (WT) and GC-C deficient mice to acute liver injury with a single injection of the hepatotoxin carbon tetrachloride. Changes in the level of expression of GC-C and its ligands uroguanylin and guanylin were quantified by real-time PCR. Liver morphology, and hepatocyte necrosis, apoptosis and proliferation, were examined at 1-3 days post-injury in mice on a mixed genetic background. Survival was followed for 14 days after carbon tetrachloride injection in wild type and GC-C deficient mice on both a mixed genetic background and on an inbred C57BL6/J background.ResultsGC-C deficient mice on the mixed genetic background nearly all died (median survival of 5 days) following carbon tetrachloride injection while WT littermates experienced only 35% mortality. Elevated levels of TUNEL-positive hepatocyte death on post-injury day 1, increased apoptosis on day 2, and increased areas of centrilobular necrosis on days 2 and 3, were evident in livers from GC-C null mice compared to WT. Collectively these data suggest increased hepatocyte death in the GC-C null mice in the early time period after injury. This corresponds temporally with increased expression of GC-C and its ligands guanylin and uroguanylin in post-injury WT mouse liver. The hepatocyte proliferative response to injury was the same in both genotypes. In contrast, there was no difference in survival between GC-C null and WT mice on the inbred C57BL/6 J background in response to acute liver injury.ConclusionsSignalling via GC-C promotes hepatocyte survival in vivo and is required for effective recovery from acute toxic injury to the liver in a strain-specific manner.

Comparison of histopathology and RT-qPCR amplification of guanylyl cyclase C for detection of colon cancer metastases in lymph nodes

AimsIn colorectal cancer (CRC), the presence of lymph node (LN) metastases is an important prognostic factor. Approximately 20% of patients diagnosed as having node-negative (pN0) CRC will relapse. Pathological nodal...

Abstract 1614: Guanylyl cyclase C signaling through vasodilator-stimulated phosphoprotein in lost in colon cancer

Abstract The biochemical pathway(s) induced by guanylyl cyclase C (GCC) regulates the intestinal crypt-villus axis by modulating proliferation, maturation and metabolic programming. During colorectal tumorigenesis, GCC signaling through cGMP becomes dysregulated as a consequence of the reduced expression of GCC endogenous hormones guanylin and uroguanylin. However, the significance of the loss of ligand-dependent signaling by GCC for the human disease remains unclear. Here, immunohistochemical examination of tumor specimens from patients compared to respective normal adjacent tissues revealed that the GCC signaling through vasodilator-stimulated phosphoprotein (VASP), a key regulator of actin polymerization and morphogenetic dynamics, is lost in colon cancer. Interruption of GCC signaling through VASP resulted in tumor depletion of the cGMP-dependent VASP phosphorylation species, and presumably reflected complex alterations of the normal initiation and propagation of the ligand-induced GCC signal. In vitro reconstitution of the ligand-dependent GCC signal through VASP induced a functional remodeling of human cancer cells which assumed a rounded, lamellipodia-free morphology with reduced proliferative and invasive abilities. Importantly, GCC ligand therapy in vivo restored the cGMP-dependent VASP phosphorylation circuit in intestinal tumors of APCmin/+ mice. These findings offer a previously undescribed tumor suppressor mechanism, cGMP-dependent VASP phosphorylation, with high translational potential for the diagnosis and therapy of colon cancer. Reactivation of cGMP signaling through VASP by GCC hormone replacement therapy may represent an innovative paradigm for the treatment of colorectal cancer in patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1614.

Linaclotide is a potent and selective guanylate cyclase C agonist that elicits pharmacological effects locally in the gastrointestinal tract

Aims Linaclotide is an orally administered 14-amino acid peptide being developed for the treatment of constipation-predominant irritable bowel syndrome (IBS-C) and chronic constipation. We determined the stability of linaclotide in the intestine, measured the oral bioavailability, and investigated whether the pharmacodynamic effects elicited in rodent models of gastrointestinal function are mechanistically linked to the activation of intestinal guanylate cyclase C (GC-C). Main methods Linaclotide binding to intestinal mucosal membranes was assessed in competitive binding assays. Stability and oral bioavailability of linaclotide were measured in small intestinal fluid and serum, respectively, and models of gastrointestinal function were conducted using wild type (wt) and GC-C null mice. Key findings Linaclotide inhibited in vitro [ 125I]-STa binding to intestinal mucosal membranes from wt mice in a concentration-dependent manner. In contrast, [ 125I]-STa binding to these membranes from GC-C null mice was significantly decreased. After incubation in vitro in jejunal fluid for 30 min, linaclotide was completely degraded. Pharmacokinetic analysis showed very low oral bioavailability (0.10%). In intestinal secretion and transit models, linaclotide exhibited significant pharmacological effects in wt, but not in GC-C null mice: induction of increased fluid secretion into surgically ligated jejunal loops was accompanied by the secretion of elevated levels of cyclic guanosine-3′,5′-monophosphate and accelerated gastrointestinal transit. Significance Linaclotide is a potent and selective GC-C agonist that elicits pharmacological effects locally in the gastrointestinal tract. This pharmacological profile suggests that orally administered linaclotide may be capable of improving the abdominal symptoms and bowel habits of patients suffering from IBS-C and chronic constipation.

Analytical Performance of a qRT-PCR Assay to Detect Guanylyl Cyclase C in FFPE Lymph Nodes of Patients With Colon Cancer

Up to 30% of patients with stage II (pN0) colon cancer develop recurrences, suggesting that the presence of lymph node (LN) metastases escaped detection at histopathologic staging. A simple way to overcome this limitation and to improve staging accuracy is to use reverse transcription-polymerase chain reaction (RT-PCR) to examine a larger fraction or an entire specimen. The Guanylyl cyclase C (GCC) gene is uniquely expressed in apical cells of the gastrointestinal tract. Its expression in colon cancer cells and metastases is conserved. Therefore, detection of GCC mRNA in LNs has been shown to be indicative of the presence of colon cancer metastases. As the current processing of LNs involves formalin fixation and paraffin embedding, we developed a method for extracting RNA from formalin-fixed paraffin-embedded LN specimens and detecting GCC mRNA by quantitative RT-PCR. The assay has a dynamic range of 5 logs, an average amplification efficiency of 98.4% (95% confidence interval, 96.6-100.3), a reaction linearity of 0.998 (95% confidence interval, 0.997-0.999), and also intraplate and interplate CVs of <1% and <5%, respectively. The test specificity was 98% with LNs collected from patients affected by conditions other than colon cancer (n=380). Sensitivity was 97% for patients with stage III colon cancer (n=34), whereas 35% of patients with stages I and II disease (n=51) had at least 1 GCC mRNA-positive LN. The high specificity of GCC mRNA suggests that routine utilization of the quantitative RT-PCR test has the potential to improve the detection of colon cancer metastases in LNs.

Linaclotide - a secretagogue and antihyperalgesic agent - what next?

Ongoing clinical trials suggest that linaclotide, a first-in-class, 14-amino acid peptide guanylate cyclase-C (GC-C) receptor agonist and intestinal secretagogue is an effective treatment for chronic constipation. A study in this issue of the Journal suggests that linaclotide also has antihyperalgesic effects in three common rat models of inflammation- and stress-induced hypersensitivity (i.e., acute trinitrobenzene sulfonic acid colitis, water avoidance stress [WAS], and restraint-induced stress) but not in naïve animals. In mice, linaclotide at least partly reduces hyperalgesia via GC-C receptors. Dose-effect relationships of linaclotide were complicated and non-linear. This viewpoint discusses human clinical trials with linaclotide and the results of this study. Potential mechanisms and clinical significance of these findings are explored. Collectively, these data suggest that GC-C receptors exert other, as yet poorly understood, effects on gastrointestinal sensitivity in conditions associated with inflammation and/or stress-induced increased intestinal permeability. However, the data need to be confirmed in humans and in long-term animal models. Further studies are also necessary to elucidate the mechanisms as these effects cannot be explained by linaclotide's known effects on epithelial GC-C receptors.

This Month in Gastroenterology

This Month in Gastroenterology

Diagnosis and treatment of guanylyl cyclase-C and colorectal cancer

Guanylyl cyelase-C (GC-C) is recently reported as the intestinal specific polypeptide receptor,which is expressed selectively by cells from intestinal mucosa,including normal intestinal cells,primary and metastatic colorectal cancer cells.Stimulated by heat-stable enterotoxin (STa),guanylin and uroguanylin,GC-C could not only regulate the proliferation and differentiation of intestinal cells,but also inhibit the proliferation of colorectal cancer cells and DNA synthesis.Micrometastases of the colorectal cancer can be found in lymph nodes,peripheral blood and liver lobes by GC-C detection.We can also perform GC-C and ligands for targeted prevention and therapy of colorectal cancer.GCC play important roles in detecting occult micrometastasos of colorectal cancer and targeted therapy. Key words: Guanylate cyclase; Colorectal neoplasms; Neoplasm metastasis; Diagnosis; Therapy

Efficacy of Linaclotide for Patients With Chronic Constipation

Linaclotide is a minimally absorbed peptide agonist of the guanylate cyclase-C receptor that stimulates intestinal fluid secretion and transit and reduces pain in animal models. We assessed the safety and efficacy of a range of linaclotide doses in patients with chronic constipation. We performed a multicenter, double-blind, placebo-controlled, parallel-group study of 310 patients with chronic constipation. Patients were randomly assigned to groups given 75, 150, 300, or 600 microg oral linaclotide or placebo once daily for 4 weeks. Symptom assessments included spontaneous bowel movements (SBMs), complete SBMs, stool consistency, straining, abdominal discomfort, and bloating. Severity of constipation, adequate relief of constipation, global relief of constipation, treatment satisfaction, quality of life, adverse events, clinical laboratory data, and electrocardiogram results were assessed. All doses of linaclotide improved the weekly rate of SBM (primary end point) compared with placebo; the increases in overall weekly number of SBMs from baseline were 2.6, 3.3, 3.6, and 4.3 for linaclotide doses of 75, 150, 300, and 600 microg, respectively, compared with 1.5 for placebo (P < or = .05 for each pair-wise comparison of a linaclotide dose to placebo). Likewise, linaclotide significantly improved the weekly rate of complete SBM, stool consistency, straining, abdominal discomfort, bloating, global assessments, and quality of life. The most common and only dose-related adverse event was diarrhea (only 6 patients discontinued treatment because of diarrhea). Linaclotide therapy was associated with few adverse events and produced rapid and sustained improvement of bowel habits, abdominal symptoms, global relief, and quality of life in patients with chronic constipation.

Receptor guanylyl cyclase C (GC-C): regulation and signal transduction

Receptor guanylyl cyclase C (GC-C) is the target for the gastrointestinal hormones, guanylin, and uroguanylin as well as the bacterial heat-stable enterotoxins. The major site of expression of GC-C is in the gastrointestinal tract, although this receptor and its ligands play a role in ion secretion in other tissues as well. GC-C shares the domain organization seen in other members of the family of receptor guanylyl cyclases, though subtle differences highlight some of the unique features of GC-C. Gene knock outs in mice for GC-C or its ligands do not lead to embryonic lethality, but modulate responses of these mice to stable toxin peptides, dietary intake of salts, and development and differentiation of intestinal cells. It is clear that there is much to learn in future about the role of this evolutionarily conserved receptor, and its properties in intestinal and extra-intestinal tissues.

Can Colorectal Cancer be Prevented or Treated by Oral Hormone Replacement Therapy?

Guanylyl cyclase C (GCC) is the receptor specifically expressed by intestinal cells for the paracrine hormones guanylin and uroguanylin and diarrheagenic bacterial heat-stable enterotoxins. This tissue-specific receptor coordinates lineage-dependent regulation of epithelial homeostasis, and its disruption contributes to intestinal tumorigenesis. It coordinates regenerative and metabolic circuits by restricting the cell cycle and proliferation and programming metabolic transitions central to organizing the dynamic crypt-surface axis. Further, mice deficient in GCC signaling are more susceptible to colon cancer induced by Apc mutations or the carcinogen azoxymethane. Moreover, guanylin and uroguanylin are gene products most commonly lost, early, in colon cancer in animals and humans. The role of GCC as a tumor suppressing receptor regulating proliferation and metabolism, together with the universal loss of guanylin and uroguanylin in tumorigenesis, suggests a model in which colorectal cancer is a paracrine hormone deficiency syndrome. In that context, activation of GCC reverses the tumorigenic phenotype by limiting growth of colorectal cancer cells by restricting progression through the G1/S transition and reprogramming metabolic circuits from glycolysis to oxidative phosphorylation, limiting bioenergetic support for rapid proliferation. These observations suggest a pathophysiological hypothesis in which GCC is a lineage-dependent tumor suppressing receptor coordinating proliferative homeostasis whose dysregulation through hormone loss contributes to neoplasia. The correlative therapeutic hypothesis suggests that colorectal cancer is a disease of hormone insufficiency that can be prevented or treated by oral supplementation with GCC ligands.

Ectopic expression of guanylyl cyclase C in gastric cancer as a potential biomarker and therapeutic target

To investigate the expression of guanylyl cyclase C (GCC) in human gastric cancer (GC) tissues and assess the effect of GCC small interfering RNA (siRNA) on the proliferation and apoptosis of SGC-7901. The expression of GCC in 30 specimens and three human GC cell lines (SGC-7901, AGS, NCI-N87) were detected by RT-PCR for messenger RNA (mRNA) by Western blot and immunofluorescence for proteins. Recombinant plasmids containing GCC siRNA and scrambled siRNA were constructed and transfected into SGC-7901 cells, respectively. A cell counting kit-8, flow cytometry (FCM) and terminal deoxynucleotidyl transferase (TDT)-mediated dUTP-biotin nick end-labeling were used to evaluate cell viability, cell cycle distribution and apoptosis, followed by wound healing assay and cell adherent assay for cell motility and adherent, respectively. The expression of GCC was absent in paracancerous tissues, whereas the GCC mRNA and protein expressions were detected in 20/30 and 19/30 of GC specimens, respectively. Moreover, intestinal GC was statistically different from diffuse GC (P < 0.05). The proliferation of SGC-7901 cells was markedly inhibited by GCC siRNA-3 (P < 0.05) and cell morphological changes including volumetric reduction, karyopyknosis and karyorrhexis were observed. FCM showed that the cell count in the sub-G0/G1 peak increased from 5.47% (48 h after transfection) to 5.63% (72 h after transfection). The wound healing assay and cell adherent assay revealed that GCC gene silencing decreased cell motility and adherent. The over-expression of GCC has been detected in intestinal type GC. GCC siRNA can effectively inhibit the proliferation and invasion of SGC-7901 cells and induce cell apoptosis. GCC might be a novel biomarker and therapeutic target for GC.

Cross Talk between Receptor Guanylyl Cyclase C and c-src Tyrosine Kinase Regulates Colon Cancer Cell Cytostasis

Increased activation of c-src seen in colorectal cancer is an indicator of a poor clinical prognosis, suggesting that identification of downstream effectors of c-src may lead to new avenues of therapy. Guanylyl cyclase C (GC-C) is a receptor for the gastrointestinal hormones guanylin and uroguanylin and the bacterial heat-stable enterotoxin. Though activation of GC-C by its ligands elevates intracellular cyclic GMP (cGMP) levels and inhibits cell proliferation, its persistent expression in colorectal carcinomas and occult metastases makes it a marker for malignancy. We show here that GC-C is a substrate for inhibitory phosphorylation by c-src, resulting in reduced ligand-mediated cGMP production. Consequently, active c-src in colonic cells can overcome GC-C-mediated control of the cell cycle. Furthermore, docking of the c-src SH2 domain to phosphorylated GC-C results in colocalization and further activation of c-src. We therefore propose a novel feed-forward mechanism of activation of c-src that is induced by cross talk between a receptor GC and a tyrosine kinase. Our findings have important implications in understanding the molecular mechanisms involved in the progression and treatment of colorectal cancer.

Guanylate cyclase C-mediated antinociceptive effects of linaclotide in rodent models of visceral pain

BACKGROUND Linaclotide is a novel, orally administered investigational drug currently in clinical development for the treatment of constipation-predominant irritable bowel syndrome (IBS-C) and chronic idiopathic constipation. Visceral hyperalgesia is a major pathophysiological mechanism in IBS-C. Therefore, we investigated the anti-nociceptive properties of linaclotide in rodent models of inflammatory and non-inflammatory visceral pain and determined whether these pharmacological effects are linked to the activation of guanylate cyclase C (GC-C). METHODS Orally administered linaclotide was evaluated in non-inflammatory acute partial restraint stress (PRS) and acute water avoidance stress (WAS) models in Wistar rats, and in a trinitrobenzene sulfonic acid (TNBS)-induced inflammatory model in Wistar rats and GC-C null mice. KEY RESULTS In TNBS-induced colonic allodynia, linaclotide significantly decreased the number of abdominal contractions in response to colorectal distension without affecting the colonic wall elasticity change in response to distending pressures after TNBS. However, linaclotide had no effect on visceral sensitivity under basal conditions. In addition, linaclotide significantly decreased colonic hypersensitivity in the PRS and WAS models. In wild type (wt) and GC-C null mice, the instillation of TNBS induced similar hyperalgesia and allodynia. However, in post-inflammatory conditions linaclotide significantly reduced hypersensitivity only in wt mice, but not in GC-C null mice. CONCLUSIONS & INFERENCES These findings indicate that linaclotide has potent anti-nociceptive effects in several mechanistically different rodent models of visceral hypersensitivity and that these pharmacological properties of linaclotide are exerted through the activation of the GC-C receptor. Therefore, linaclotide may be capable of decreasing abdominal pain in patients suffering from IBS-C.

Guanylyl cyclase C is a specific marker for differentiating primary and metastatic ovarian mucinous neoplasms

The aim was to assess the value of GCC in distinguishing primary ovarian mucinous neoplasms from metastatic mucinous adenocarcinomas with ovarian involvement. Guanylyl cyclase C (GCC) is a brush border membrane receptor for the endogenous peptides guanylin and uroguanylin, and the homologous diarrhoeagenic bacterial heat-stable enterotoxins that is selectively expressed by epithelial cells from the duodenum to the rectum, but not by normal epithelia of the stomach or oesophagus, or normal extramucosal cells in humans. Fifty ovarian tumours: 27 primary ovarian mucinous neoplasms (seven cystadenomas, 10 borderline tumours and 10 cystadenocarcinomas) and 23 metastatic mucinous adenocarcinomas with ovarian involvement [13 colorectal adenocarcinomas, four gastric adenocarcinomas, six appendiceal mucinous tumours (four adenocarcinomas, one with neuroendocrine features, and two appendiceal mucinous cystadenomas)] were studied. For primary ovarian mucinous neoplasms, 25 of 27 were negative for GCC. Twelve of 13 cases of colorectal adenocarcinoma (except for one neuroendocrine adenocarcinoma) were positive for GCC. Three of four appendiceal mucinous adenocarcinomas were positive for GCC in both the primary and metastatic tumours (except for one neuroendocrine adenocarcinoma). Two of two appendiceal mucinous cystadenomas were positive for GCC. Of four cases of gastric adenocarcinoma with ovarian involvement, only one (primary tumour) exhibited focal GCC staining. GCC is a useful marker for differentiating between primary and secondary ovarian mucinous neoplasms.

Guanylyl Cyclase C in Colorectal Cancer: Susceptibility Gene and Potential Therapeutic Target

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.

M1696 Novel Role of Guanylyl Cyclase C (GCC) in Maintenance of Intestinal Barrier Function

M1696 Novel Role of Guanylyl Cyclase C (GCC) in Maintenance of Intestinal Barrier Function

Guanylyl Cyclase C Prevents Colon Cancer Metastasis by Regulating Tumor Epithelial Cell Matrix Metalloproteinase-9

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.