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Detection of guanylyl cyclase C mRNA in lymph nodes of resected stage II colorectal cancer is highly correlated with the risk of tumor recurrence. If validated, these results could have significant implications for the selection of patients for adjuvant therapy in this disease. Clin Cancer Res; 17(10); 3053–5. ©2011 AACR.

Purpose: Lymph node involvement by histopathology informs colorectal cancer prognosis, whereas recurrence in 25% of node-negative patients suggests the presence of occult metastasis. GUCY2C (guanylyl cyclase C) is a marker of colorectal cancer cells that identifies occult nodal metastases associated with recurrence risk. Here, we defined the association of occult tumor burden, quantified by GUCY2C reverse transcriptase-PCR (RT-PCR), with outcomes in colorectal cancer. Experimental Design: Lymph nodes (range: 2–159) from 291 prospectively enrolled node-negative colorectal cancer patients were analyzed by histopathology and GUCY2C quantitative RT-PCR. Participants were followed for a median of 24 months (range: 2–63). Time to recurrence and disease-free survival served as primary and secondary outcomes, respectively. Association of outcomes with prognostic markers, including molecular tumor burden, was estimated by recursive partitioning and Cox models. Results: In this cohort, 176 (60%) patients exhibited low tumor burden (MolLow), and all but four remained free of disease [recurrence rate 2.3% (95% CI, 0.1–4.5%)]. Also, 90 (31%) patients exhibited intermediate tumor burden (MolInt) and 30 [33.3% (23.7–44.1)] developed recurrent disease. Furthermore, 25 (9%) patients exhibited high tumor burden (MolHigh) and 17 [68.0% (46.5–85.1)] developed recurrent disease (P < 0.001). Occult tumor burden was an independent marker of prognosis. MolInt and MolHigh patients exhibited a graded risk of earlier time to recurrence [MolInt, adjusted HR 25.52 (11.08–143.18); P < 0.001; MolHigh, 65.38 (39.01–676.94); P < 0.001] and reduced disease-free survival [MolInt, 9.77 (6.26–87.26); P < 0.001; MolHigh, 22.97 (21.59–316.16); P < 0.001]. Conclusion: Molecular tumor burden in lymph nodes is independently associated with time to recurrence and disease-free survival in patients with node-negative colorectal cancer. Clin Cancer Res; 17(10); 3293–303. ©2011 AACR.

Tumor cells in regional lymph nodes are a key prognostic marker of survival and predictive marker of response to adjuvant chemotherapy in colorectal cancer. However, clinicopathologic techniques to detect lymph node metastases remain imperfect, and approximately 30% of patients with lymph nodes negative by histology (pN0) develop recurrent disease, reflecting occult metastases that escape detection. These observations underscore an unmet clinical need for accurate approaches to identify occult nodal metastases in colorectal cancer patients. GUCY2C is a receptor whose expression normally is restricted to intestinal epithelial cells, but is universally overexpressed by colorectal cancer cells. A prospective, multicenter, blinded clinical trial established the prognostic utility of GUCY2C qRT-PCR to detect occult nodal metastases in pN0 colorectal cancer patients. Molecular staging revealed that approximately 13% of pN0 patients were free of cancer cells, while approximately 87% had GUCY2C results that suggested occult metastases. The presence of occult nodal metastases was the most powerful independent predictor of time to recurrence and disease-free survival. These observations establish the utility of molecular detection of occult nodal metastases for assessing prognostic risk in pN0 colorectal cancer patients. Advancing GUCY2C into staging paradigms in clinical laboratories will require validation in independent patient populations, definition of the relationship between the quantity of occult tumor metastases and risk, and determination of the utility of GUCY2C qRT-PCR to identify pN0 patients who might benefit from adjuvant chemotherapy.

Proteins binding Escherichia coli heat-stable enterotoxin were isolated from the cytoskeleton of intestinal membranes using an affinity matrix of biotinylated ST immobilized on monomeric avidin-agarose. ST binding proteins were purified 343-fold using this affinity technique, with 7% of the initial binding activity recovered in these preparations. ST binding proteins isolated by affinity chromatography possessed a native and subunit molecular mass of 56 kDa. These preparations exhibited both high- and low-affinity binding sites for ST. Guanylate cyclase in extracts of the intestinal membrane cytoskeleton was completely recovered in fractions which did not associate with the affinity matrix. In addition, ST binding proteins isolated by affinity chromatography were devoid of guanylate cyclase activity. These data, taken together with those obtained previously with crude and partially purified receptors, suggest that ST binds to different proteins in intestinal membranes, some of which do not possess guanylate cyclase activity.

A fragment of guanylate cyclase C (GC-C) of about 1.7 k bp corresponding to amino acids 1-553 spanning the extracellular and transmembrane domains and a portion of the intracellular region was amplified using template cDNA prepared from rat intestinal cells by the polymerase chain reaction method. The cloned 1.7 k bp fragment was inserted into the mammalian expression vector pCGUT and the truncated GC-C expressed on the surface of COS-7 cells was demonstrated to bind heat-stable enterotoxin by photo affinity labeling with 125 I- N -5-azidonitrobenzoyl-STh[5-19]. Analysis by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis showed that the truncated GC-C formed dimers on the surface of COS-7 cells. The intracellular region of GC-C was found not to be necessary for dimer formation by the GC-C. Comparison of the molecular weights of the truncated GC-C expressed in COS-7 cells and Escherichia coli suggested that the truncated GC-C was glycosylated in the mammalian expression system.

An important approach to the major health problem of bacterial infection in young children has been to examine bacterial toxin binding to microvillus membrane receptors, the signal transduction produced by that interaction and the mechanisms of fluid secretion in the developing intestine as a basis for toxigenic diarrhea in the infant population. These studies indicate that receptor binding and effector responses may be subjected to developmental regulation. This regulation process of toxin interaction with the developing intestine may have an enhanced or harmful effect or, under some circumstances, may have a beneficial effect and be protective to the vulnerable child. Specific mechanisms for the developmental control of receptor expression may involve the regulation of individual glycosyltransferases responsible for the addition of receptor sugar sequences to glycolipids and/or glycoproteins, presumably at the transcriptional level. Furthermore, although highly speculative at this point, the differential expression of signal transducers (e.g., guanine nucleotide-regulatory proteins or G proteins) and ion transporters (e.g., Na+,K(+)-stimulated adenosine triphosphatase, the Cl- channels, etc.) during development may also alter the neonatal host's responsiveness. Therefore, the developmental control of microvillus membrane receptors, signal transduction mechanisms, and ion transport systems in the gastro-intestinal tract may in part contribute to the altered host sensitivity in toxigenic diarrhea of infancy.

Enterotoxigenic Escherichia coli elaborate a heat-stable enterotoxin that causes diarrhea in humans and animals. The primary event in the diarrheal cascade is the binding of this enterotoxin to specific receptors on enterocytes and activation of guanylyl cyclase. Two intestinal cell lines, Caco-2 and IEC-6, were tested for the presence of these receptors. Although both cell lines exhibited specific binding, only the Caco-2 cell line responded to heat-stable enterotoxin with increased guanylyl cyclase activity. Cloning and expression studies confirmed that the receptor present in Caco-2 cells is a homologue of guanylyl cyclase C, a known transmembrane heat-stable enterotoxin receptor. Expression of the receptor in differentiating Caco-2 cells increases with cell maturation, indicating that these cells are a suitable model for future studies. However, Northern and polymerase chain reaction analyses demonstrated that guanylyl cyclase C is not expressed in IEC-6 cells, strongly suggesting the presence of a novel heat-stable enterotoxin receptor that is not coupled to guanylyl cyclase activity.

Cyclic GMP is a ubiquitous second messenger that regulates a wide array of physiologic processes such as blood pressure, long bone growth, intestinal fluid secretion, phototransduction and lipolysis. Soluble and single-membrane-spanning enzymes called guanylyl cyclases (GC) synthesize cGMP. In humans, the latter group consists of GC-A, GC-B, GC-C, GC-E and GC-F, which are also known as NPR-A, NPR-B, StaR, Ret1-GC and Ret2-GC, respectively. Membrane GCs are activated by peptide ligands such as atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), C-type natriuretic peptide (CNP), guanylin, uroguanylin, heat stable enterotoxin and GC-activating proteins. Nesiritide and carperitide are clinically approved peptide-based drugs that activate GC-A. CD-NP is an experimental heart failure drug that primarily activates GC-B but also activates GC-A at high concentrations and is resistant to degradation. Inactivating mutations in GC-B cause acromesomelic dysplasia type Maroteaux dwarfism and chromosomal mutations that increase CNP concentrations are associated with Marfanoid-like skeletal overgrowth. Pump-based CNP infusions increase skeletal growth in a mouse model of the most common type of human dwarfism, which supports CNP/GC-B-based therapies for short stature diseases. Linaclotide is a peptide activator of GC-C that stimulates intestinal motility and is in late-stage clinical trials for the treatment of chronic constipation. This review discusses the discovery of cGMP, guanylyl cyclases, the general characteristics and therapeutic applications of GC-A, GC-B and GC-C, and emphasizes the regulation of transmembrane guanylyl cyclases by phosphorylation and ATP.

The peptide uroguanylin (Ugn) regulates enteric and renal electrolyte transport. Previous studies have shown that Ugn and its receptor GC-C (a ligand-activated guanylate cyclase) are abundant in the intestine. Less is known about Ugn and GC-C expression in the kidney. Here, we identify a 9.4-kDa polypeptide in rat kidney extracts that appears, based on its biochemical and immunological properties, to be authentic prouroguanylin (proUgn). This propeptide is relatively plentiful in the kidney (∼16% of intestinal levels), whereas its mRNA is marginally present (

Colon cancer, the second leading cause of cancer-related mortality worldwide, originates from the malignant transformation of intestinal epithelial cells. The intestinal epithelium undergoes a highly organized process of rapid regeneration along the crypt-villus axis, characterized by proliferation, migration, differentiation and apoptosis, whose coordination is essential to maintaining the mucosal barrier. Disruption of these homeostatic processes predisposes cells to mutations in tumor suppressors or oncogenes, whose dysfunction provides transformed cells an evolutionary growth advantage. While sequences of genetic mutations at different stages along the neoplastic continuum have been established, little is known of the events initiating tumorigenesis prior to adenomatous polyposis coli (APC) mutations. Here, we examine a role for the corruption of homeostasis induced by silencing novel tumor suppressors, including the intestine-specific transcription factor CDX2 and its gene target guanylyl cyclase C (GCC), as early events predisposing cells to mutations in APC and other sequential genes that initiate colorectal cancer. CDX2 and GCC maintain homeostatic regeneration in the intestine by restricting cell proliferation, promoting cell maturation and adhesion, regulating cell migration and defending the intestinal barrier and genomic integrity. Elimination of CDX2 or GCC promotes intestinal tumor initiation and growth in aged mice, mice carrying APC mutations or mice exposed to carcinogens. The roles of CDX2 and GCC in suppressing intestinal tumorigenesis, universal disruption in their signaling through silencing of hormones driving GCC, and the uniform overexpression of GCC by tumors underscore the potential value of oral replacement with GCC ligands as targeted prevention and therapy for colorectal cancer.

Aims In 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 stage misclassification due to sampling error resulting from the small volume of tissue tested has been proposed to explain this recurrence rate in pN0 patients. The authors compared the assessment of node positivity by histopathology (HP) with a molecular method which can accommodate larger tissue volumes. Methods Detection rate of guanylyl cyclase C (GCC) mRNA was determined in 1495 LNs from 99 CRC patients. Using a subset of 647 LNs, multiple levels of HP analysis were compared with GCC mRNA molecular detection. Finally, clinicopathological factors were correlated with the molecular detection of GCC and clinical outcome in 123 patients with pN0 colon cancer. Results GCC mRNA was detected in 8.0% of the 560 nodes initially identified as HP-negative, whereas two repeat HP examinations detected 3.0% of these cases. In HP-positive LNs, the GCC mRNA detection rate was 90% (78/87) when half-LN were tested. Testing the entire LN remaining after HP by GCC increased the detection rate of HP-positive LNs to 95% (p=0.027). In comparison, 75% (65/87) and 92% (80/87) of the LN positive by clinical HP remained positive when one or two subsequent sections were examined by HP. Finally, patients with pN0 disease who were GCC-positive exhibited an earlier time of recurrence (hazard ratio, 3.54; 95% CI 1.40 to 8.98; p=0.0077). Conclusions Molecular detection of tumour cells in LNs may have prognostic value in identifying patients diagnosed as having pN0 colon cancer who will relapse following surgery.

While bile acids are a risk factor for tumorigenesis induced by reflux disease, the mechanisms by which they contribute to neoplasia remain undefined. Here, we reveal that in gastroesophageal junction (GEJ) cells bile acids activate a tissue-specific developmental program defining the intestinal epithelial cell phenotype characterizing GEJ metaplasia. Deoxycholic acid (DCA) inhibited phosphorylation of EGF receptors (EGFRs) suppressing the proto-oncogene AKT. Suppression of EGFRs and AKT by DCA actuated an intestine-specific cascade in which NF-kappaB transactivated the tissue-specific transcription factor CDX2. In turn, CDX2 orchestrated a lineage-specific differentiation program encompassing genes characterizing intestinal epithelial cells. Conversely, progression from metaplasia to invasive carcinoma in patients, universally associated with autonomous activation of EGFRs and/or AKT, was coupled with loss of this intestinal program. Thus, bile acids induce intestinal metaplasia at the GEJ by activating the lineage-specific differentiation program involving suppression of EGFR and AKT, activating the NF-kappaB-CDX2 axis. Induction of this axis provides the context for lineage-addicted tumorigenesis, in which autonomous activation of AKT corrupts adaptive intestinal NF-kappaB signaling, amplifying tumorigenic programs.

Linaclotide is a first-in-class, orally administered 14-amino acid peptide that is in development for the treatment of irritable bowel syndrome with constipation and chronic constipation. We have characterized the solution structure of linaclotide, the in vitro binding and agonist activity to guanylate cyclase C receptors, the stability of linaclotide under conditions mimicking the gastric environment, oral bioavailability, and the pharmacodynamic effects in rat models of gastrointestinal transit and intestinal secretion. Nuclear magnetic resonance spectroscopy analysis determined that the molecular structure of linaclotide is stabilized by three intramolecular disulfide bridges. Linaclotide exhibited high affinity and pH-independent binding (K(i): 1.23-1.64 nM) to guanylate cyclase C receptors on human colon carcinoma T84 cells and concomitantly, linaclotide binding resulted in a significant, concentration-dependent accumulation of intracellular cyclic guanosine-3', 5'-monophosphate (cGMP) (EC₅₀:99 nM). Linaclotide was stable after 3 h incubation in simulated gastric fluid (pH 1) and similarly, was completely resistant to hydrolysis by pepsin. Pharmacokinetic analysis of linaclotide showed very low oral bioavailability (0.1%). Orally administered linaclotide elicited a significant, dose-dependent increase in gastrointestinal transit rates in rats at doses of ≥5 μg/kg. Exposure of surgically ligated small intestinal loops to linaclotide induced a significant increase in fluid secretion, accompanied by a significant increase in intraluminal cGMP levels. These results suggest that the guanylate cyclase C agonist linaclotide elicits potent pharmacological responses locally in the gastrointestinal tract, and that orally administered guanylate cyclase C agonists may be capable of improving bowel habits in patients suffering from irritable bowel syndrome with constipation and chronic constipation.

Heat-stable enterotoxin (STa) secretion from Enterotoxigenic Escherichia coli (ETEC) is crucial for the pathogenesis of diarrhea in both animal and human. The goal of this study was to investigate the distribution of the STa-specific receptors in the newborn camel’s enterocytes and brush border membrane vesicles (BBMVs). Flow cytometric analysis was used to investigate the density of STa-receptors on enterocytes and BBMVs prepared from anterior jejunum, posterior jejunum, ileum, and colon. Strong density and affinity of STa-receptors was present on enterocytes and BBMVs of the ileum compared to that in the other intestinal segments. It was concluded that the ileum is the major target for STa action in newborn camels.

Polymorphisms in genes that influence the expression of toxin receptors could contribute to Sudden Infant Death Syndrome (SIDS) and unexplained Sudden Unexpected Death in Infancy (uSUDI) for which there is evidence of toxin involvement. We aimed to determine whether TCRBV3S1 allele 2 could be involved in a staphylococcal toxic shock hypothesis for uSUDI. Observed frequencies of the TCRBV3S1*2 allele and genotype in 48 Australian uSUDI cases and 96 live comparison infants did not differ. In future the role of other toxin receptor gene polymorphisms deserves investigation.

Escherichia coli heat-stable enterotoxin (STa) mediates diarrheal disease by binding to and activating an intestinal transmembrane receptor, guanylate cyclase C (GC-C). To test the hypotheses that there was 1) increased perinatal expression of GC-C in rat intestine and 2) GC-C expression and STa binding in extraintestinal tissues of immature rat, we prepared whole cell membranes and total RNA from jejunum, ileum, colon, liver, kidney, heart, lung, brain, testis, and placenta of rats ranging in age from 12 days gestation to adult. Northern analysis demonstrated the presence of a unique 3.8-kb mRNA transcript at all ages in the jejunum, ileum, colon, and, to a lesser degree, in the testis. GC-C was also detected by Northern analysis in liver (from gestational age 18 days through 14 days postnatal) and in placenta. Steady-state mRNA encoding GC-C was not detected by Northern analysis in the other organs examined. GC-C-specific mRNA expression was greatest in the perinatal period in the jejunum, ileum, and liver. Specific binding of 125I-labeled STa was found in each of the tissue membranes in which GC-C mRNA was present; binding was not present in those tissues that had no detectable GC-C mRNA. The existence of GC-C in extraintestinal organs in the rat, and the development changes in GC-C expression support our hypothesis that GC-C, apart from its role as an STa receptor in mediating diarrheal disease, also serves as a receptor for an endogenous ligand.

Staphylococcal toxic shock syndrome toxin-1 (TSST-1) as well as staphylococcal enterotoxin A (SEA) and B (SEB) have recently been shown to bind directly to the class II major histocompatibility antigen, HLA-DR. Whereas others have characterized TSST-1 and SEA binding to HLA-DR on transfected L cells or B lymphoma cell lines, we sought evidence for direct binding of TSST-1 and SEA to HLA-DR on purified human monocytes. A single class of high-affinity receptors was found for both TSST-1 (dissociation constant (Kd) 40 nM, 3.4 × 104 receptors per cell) and SEA (Kd 12 nM, 3.2 × 104 receptors per cell) on normal human monocytes. Affinity cross-linking of 125I-labeled toxins to monocytes revealed the presence of two membrane protein subunits with molecular masses consistent with the α and β chains of human HLA-DR (35 and 28 kDa, respectively). The anti-HLA-DR monoclonal antibody L243, but not L203 or 2.06, inhibited radiolabeled toxin binding to human monocytes and neutralized the mitogenic response of human T lymphocytes to both toxins. However, L243 was consistently more effective in blocking radiolabeled TSST-1 than SEA binding to human monocytes from the same donors, suggesting that TSST-1 and SEA may be binding to overlapping epitopes rather than to the same epitope on HLA-DR. Because TSST-1 and SEB bind to distinct epitopes on HLA-DR and because SEA cross competes with both TSST-1 and SEB on the HLA-DR receptor, we postulate that SEA occupies a binding site within HLA-DR that overlaps both TSST-1 and SEB. Future studies focused on receptor-mediated binding of these toxins to human monocytes and T lymphocytes from normal donors and toxic shock syndrome patients may reveal the underlying anomalies that predispose particular individuals to toxic shock syndrome. Key words: monocytes, staphylococcal toxic shock syndrome toxin-1, receptors, HLA-DR, staphylococcal enterotoxin A.

Binding of Escherichia coli heat-stable enterotoxin to its receptor is critical to the initiation of toxin-induced secretion and diarrheal disease; it is also likely, however, that this receptor binds an endogenous ligand. In order to characterize the expression of the heat-stable enterotoxin receptor in the small intestine, we isolated epithelial cells from villus tip to crypt in rat jejunum and ileum. Binding of radiolabeled toxin was maximal in the villus preparations and gradually decreased along the villus-to-crypt axis, paralleling the decline of sucrase activity. Northern blots of total RNA identified a single heat stable enterotoxin receptor transcript (3.8 kb), predominantly in the villus cell fractions. In situ hybridization demonstrated clear signal in the villus cells with no apparent signal in the crypt cells, lamina propria or muscularis. Expression of this receptor was greatest after enterocytes leave the proliferative cycle and enter villi. This pattern of gene and protein expression may reflect a role of this receptor in binding endogenous ligands which in turn may regulate intestinal ion flux along the villus-to-crypt axis.

Undernutrition in human infants is associated with more prolonged episodes of diarrheal disease. Therefore, we tested the hypothesis that malnutrition prolongs the duration of Escherichia coli heat-stable enterotoxin-induced rat jejunal secretion. At weaning, rats were separated into two groups: malnourished rats were fed 50% of the previous day's intake of the fully fed control group. After approximately 2 wk of pair feeding, when malnourished rats weighed less than or equal to 60% of the full fed control group, we measured the secretory response to heat-stable enterotoxin in ligated jejunal loops. Toxin-induced secretion was equal in both groups until 30 min incubation time, after which net secretion continued to increase in the malnourished group but decreased in the fully fed group. Jejunal brush border membranes prepared from malnourished and fully fed rats demonstrated similar heat-stable enterotoxin receptor density, avidity of binding and guanyl cyclase activation. In both groups, radiolabeled toxin injected into in situ jejunal loops was converted into an altered radioligand unable to bind to brush border membranes. However, in malnourished rats, there was both increased appearance of two additional radioligands that still retained their ability to bind to brush border membranes and persistence of biologically active unlabeled toxin as measured in the suckling mouse bioassay. Our studies demonstrate that reduced or delayed inactivation of heat-stable enterotoxin, with continued presence of active toxin species, may contribute to prolonged secretion in the jejunum of malnourished rats.

To determine the contribution of the colon in Escherichia coli heat-stable enterotoxin-mediated diarrheal disease, toxin binding, guanyl cyclase activation, and toxin-induced water flux in the rat colon and ileum were compared. Scatchard analysis suggested a single class of heat-stable enterotoxin receptors with an affinity constant of binding of 10(9) L/mol in both colonocytes and ileocytes; however, the number of toxin receptors per cell was 3.5-fold greater in coloncytes than ileocytes (8.32 +/- 1.33 x 10(5) vs. 2.33 +/- 0.28 x 10(5) receptors per cell; P = 0.02). Heat-stable enterotoxin stimulated guanyl cyclase activation in an identical dose-dependent manner in proximal colonic and ileal membranes, with similar sensitivity and maximum response. Heat-stable enterotoxin also inhibited net water flux to a similar degree in both colon and ileum (-47.8 vs. -48.4 microL.cm-1.h-1, respectively) at a dose of 8 nmol/L. At this dose in the colon, because of a higher baseline of absorption, absorption continued, but at a diminished level. At this dose in the ileum, heat-stable enterotoxin induced net secretion. These data are consistent with the concept that heat-stable enterotoxin-induced diarrheal disease results from a decreased absorptive capacity in the colon in the face of increased small intestinal fluid secretion.

Heat-stable enterotoxins (ST) activate guanylyl cyclase in T84 cells, rapidly and specifically. Activation is monitored by cGMP production and occurs at lower concentrations of ST than required for eliciting fluid accumulation in suckling mice. Atrial natriuretic factor (ANF) neither activates guanylyl cyclase nor modulates the response to ST in T84 cells, indicating the absence of receptors for ANF on T84 cells. Monitoring the production of cGMP under conditions known to alter fluid accumulation in suckling mice is an accurate and quantifiable assay of ST activity and its interaction with the receptor. STs produced by Escherichia coli, Vibrio cholerae non-01 and Yersinia enterocolitica individually produce elevated levels of cGMP in T84 cells, but to differing extents, suggesting that this model system can be used to elucidate the different events of ST-receptor interactions at the molecular level.

Active receptors for Escherichia coli heat-stable enterotoxin (ST) were partially purified by ligand-affinity chromatography. The affinity column was prepared by coupling ST to biotin derivatized with an extended N-hydroxysuccinylated spacer arm prior to binding to monomeric avidin immobilized on agarose. Detergent extracts of rat intestinal mucosa membranes were quantitatively depleted of ST binding activity when chromatographed on this affinity matrix. Biotinylated ST-receptor complexes were eluted from affinity columns with 2 mM biotin and these complexes quantitatively dissociated with bile salts. Using this technique, functional ST receptors were purified maximally about 2000-fold, with about 3% of the total activity in crude extracts recovered in these purified preparations. Analysis of affinity-purified preparations by polyacrylamide gel electrophoresis and silver staining demonstrated a major protein subunit of 74 kDa. Affinity cross-linking of these preparations to 125I-ST demonstrated specific labeling predominantly of the 74-kDa subunit. In addition, lower amounts of labeled ST were incorporated into subunits of 164 and 45 kDa, confirming the heterogeneous nature of ST receptors. Purified receptors bound ST in a concentration-dependent fashion, with an IC50 of 10(-9) M. These studies demonstrate that ligand-affinity chromatography can be employed to purify ST receptors. The availability of purified receptors will facilitate further studies of mechanisms underlying ST-induced intestinal secretion.

Colorectal cancer immunotherapy is limited by the paucity of available target antigens fulfilling the necessary criteria of tumor-specificity, sufficient immunogenicity and universal association with disease. A novel class of immune targets, cancer mucosa antigens (CMAs), whose expression normally is confined to mucosae but maintained during neoplastic transformation, promises to overcome these imitations, enjoying the advantage of immune compartmentalization, preventing autoimmune disease, while permitting therapeutic anti-tumor responses. Indeed, therapeutic immunization against the model CMA guanylyl cyclase c (GCC) extends survival in mouse models of established parenchymal colorectal cancer metastases with antitumor efficacy superior to currently available antigens. Here adjuvanation of therapeutic antitumor immunity to GCC was explored employing the cytokines IL-2 and GM-CSF in a mouse model of metastatic colorectal cancer. Combining plasmids expressing murine IL-2 or GM-CSF with recombinant viral vector immunization to GCC enhanced antitumor efficacy beyond viral vector immunization alone. These studies support the incorporation of IL-2 and GM-CSF in CMA-targeted immunization regimens for established colorectal cancer metastases.

Receptor guanylyl cyclases are multidomain proteins, and ligand binding to the extracellular domain increases the levels of intracellular cGMP. The intracellular domain of these receptors is composed of a kinase homology domain (KHD), a linker of approximately 70 amino acids, followed by the C-terminal guanylyl cyclase domain. Mechanisms by which these receptors are allosterically regulated by ligand binding to the extracellular domain and ATP binding to the KHD are not completely understood. Here we examine the role of the linker region in receptor guanylyl cyclases by a series of point mutations in receptor guanylyl cyclase C. The linker region is predicted to adopt a coiled coil structure and aid in dimerization, but we find that the effects of mutations neither follow a pattern predicted for a coiled coil peptide nor abrogate dimerization. Importantly, this region is critical for repressing the guanylyl cyclase activity of the receptor in the absence of ligand and permitting ligand-mediated activation of the cyclase domain. Mutant receptors with high basal guanylyl cyclase activity show no further activation in the presence of non-ionic detergents, suggesting that hydrophobic interactions in the basal and inactive conformation of the guanylyl cyclase domain are disrupted by mutation. Equivalent mutations in the linker region of guanylyl cyclase A also elevated the basal activity and abolished ligand- and detergent-mediated activation. We, therefore, have defined a key regulatory role for the linker region of receptor guanylyl cyclases which serves as a transducer of information from the extracellular domain via the KHD to the catalytic domain.

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.

Colorectal cancer (CRC) is the third most common cancer and a leading cause of death for both men and women in North America. The staging of the CRC at the time of diagnosis is the single most important prognostic factor in determining recurrence and survival. Until 2008, accurate evaluation of CRC stages I and II was based on examination of regional lymph nodes (LNs) under a microscope to identify cancer cells. This method can detect one cancer cell in 200 normal cells, but analyzes only a fraction of the available tissue from the LN (less than 0.1%). Up to 30% of patients assessed by traditional histopathology methods as having stage II disease (negative LNs) experience a recurrence of their cancer. Previstage GCC Colorectal Cancer Staging Test, a new molecular diagnostic test, is able to identify patients at high risk of recurrence by examining their LNs for guanylyl cyclase C (GCC). GCC is a marker found in cells lining the lumen of the gastrointestinal tract. The expression of GCC is conserved in CRC and metastatic disease. Using an ultrasensitive quantitative reverse transcription (RT)-PCR, the test interrogates a patient's LN tissues to identify GCC levels consistent with metastatic (stage III) disease. The technology employed in Previstage GCC is nearly 100,000 times more sensitive than microscopic staging methods. This molecular diagnostic test allows a more thorough examination of LNs and has an analytic sensitivity of 92% and a specificity of 98%. Such a test can be used to overcome the limitations of staging by traditional histopathology alone.

Cancer mucosa antigens are emerging as a new category of self-antigens expressed normally in immunologically privileged mucosal compartments and universally by their derivative tumors. These antigens leverage the established immunologic partitioning of systemic and mucosal compartments, limiting tolerance opposing systemic antitumor efficacy. An unresolved issue surrounding self-antigens as immunotherapeutic targets is autoimmunity following systemic immunization. In the context of cancer mucosa antigens, immune effectors to self-antigens risk amplifying mucosal inflammatory disease promoting carcinogenesis. Here, we examined the relationship between immunotherapy for systemic colon cancer metastases targeting the intestinal cancer mucosa antigen guanylyl cyclase C (GCC) and its effect on inflammatory bowel disease and carcinogenesis in mice. Immunization with GCC-expressing viral vectors opposed nascent tumor growth in mouse models of pulmonary metastasis, reflecting systemic lineage-specific tolerance characterized by CD8+, but not CD4+, T-cell or antibody responses. Responses protecting against systemic metastases spared intestinal epithelium from autoimmunity, and systemic GCC immunity did not amplify chemically induced inflammatory bowel disease. Moreover, GCC immunization failed to promote intestinal carcinogenesis induced by germ-line mutations or chronic inflammation. The established role of CD8+ T cells in antitumor efficacy, but CD4+ T cells in autoimmunity, suggests that lineage-specific responses to GCC are particularly advantageous to protect against systemic metastases without mucosal inflammation. These observations support the utility of GCC-targeted immunotherapy in patients at risk for systemic metastases, including those with inflammatory bowel disease, hereditary colorectal cancer syndromes, and sporadic colorectal cancer. [Cancer Res 2009;69(8):3537–44]

Guanylyl cyclase C, a receptor for bacterial diarrheagenic enterotoxins, is expressed selectively by intestinal epithelium and is an endogenous downstream target of CDX2. The expression of Guanylyl cyclase C is preserved throughout the adenoma/carcinoma sequence in the colorectum. Detection of Guanylyl cyclase C expression by reverse transcriptase-polymerase chain reaction is currently being validated as a technique to identify occult lymph node metastases in patients with colorectal cancer and for circulating cells in the blood for postoperative surveillance. Although Guanylyl cyclase C is widely expressed by well-differentiated colorectal cancer, its expression in poorly differentiated colorectal cancer has not been evaluated. A tissue microarray was created from 69 archival specimens including 44 poorly differentiated, 15 undifferentiated or medullary, and 10 signet ring cell colorectal carcinomas. Matched normal colonic mucosa was used as a positive control. Immunohistochemical staining for Guanylyl cyclase C and CDX2 was evaluated as positive or negative based on at least a 10% extent of staining. Of the 69 tumor samples, 75%, 47%, and 90% of the poorly differentiated, medullary, and signet ring cell tumors were positive for Guanylyl cyclase C and 75%, 40% and 90% of these subsets were positive for CDX2, respectively. There was excellent correlation between Guanylyl cyclase C and CDX2 expression on a case-per-case basis (P < .0001). There was also a statistically significant difference in the Guanylyl cyclase C staining pattern between medullary carcinomas and poorly differentiated, not otherwise specified (P = .05). Immunopositivity for Guanylyl cyclase C was greater than 95% in a separately stained microarray series of well/moderately differentiated colorectal carcinomas. In conclusion, Guanylyl cyclase C expression is lost in a quarter of poorly differentiated and half of undifferentiated colorectal carcinomas. Therefore, the utility of Guanylyl cyclase C expression as a diagnostic marker for colorectal carcinoma may be questionable in poorly differentiated colorectal neoplasms.

Context The established relationship between lymph node metastasis and prognosis in colorectal cancer suggests that recurrence in 25% of patients with lymph nodes free of tumor cells by histopathology (pN0) reflects the presence of occult metastases. Guanylyl cyclase 2C (GUCY2C) is a marker expressed by colorectal tumors that could reveal occult metastases in lymph nodes and better estimate recurrence risk. Objective To examine the association of occult lymph node metastases detected by quantifying GUCY2C messenger RNA, using the reverse transcriptase–polymerase chain reaction, with recurrence and survival in patients with colorectal cancer. Design, Setting, and Participants Prospective study of 257 patients with pN0 colorectal cancer enrolled between March 2002 and June 2007 at 9 US and Canadian centers (7 academic medical centers and 2 community hospitals) provided 2570 fresh lymph nodes measuring 5 mm or larger for histopathology and GUCY2C messenger RNA analysis. Patients were followed up for a median of 24 months (range, 2-63 months) for disease recurrence or death. Main Outcome Measures Time to recurrence (primary outcome) and disease-free survival (secondary outcome) relative to expression of GUCY2C in lymph nodes. Results Thirty-two patients (12.5%) had lymph nodes negative for GUCY2C (pN0 [mol−]), and all but 2 remained free of disease during follow-up (recurrence rate, 6.3%; 95% confidence interval [CI], 0.8%-20.8%). Conversely, 225 patients (87.5%) had lymph nodes positive for GUCY2C (pN0 [mol+]), and 47 developed recurrent disease (20.9%; 95% CI, 15.8%-26.8%) (P = .006). Multivariate analyses revealed that GUCY2C in lymph nodes was an independent marker of prognosis. Patients who were pN0 (mol+) exhibited earlier time to recurrence (adjusted hazard ratio, 4.66; 95% CI, 1.11-19.57; P = .04) and reduced disease-free survival (adjusted hazard ratio, 3.27; 95% CI, 1.15-9.29; P = .03). Conclusion Expression of GUCY2C in histologically negative lymph nodes appears to be independently associated with time to recurrence and disease-free survival in patients with pN0 colorectal cancer.

Uroguanylin (UGN) is a peptide hormone that binds to and activates the intestinal epithelial cell (IEC) transmembrane receptor guanylate cyclase C (GC-C), which in turn increases intracellular cGMP. Gene targeting of murine UGN or GC-C results in significantly lower levels of cGMP in IECs. On the basis of effects of cGMP in nonintestinal systems, we hypothesized that loss of GC-C activation would increase intestinal epithelial apoptosis following radiation-induced injury. We first compared apoptosis from the proximal jejunum of C57BL/6 wild-type (WT) and GC-C knockout (KO) mice 3 h after they received 5 Gy of γ-irradiation. We then investigated whether supplementation via intraperitoneal injection of 1 mM 8BrcGMP would mitigate radiation-induced apoptosis in these experimental animals. Identical experiments were performed in BALB/c UGN WT and KO mice. Apoptosis was assessed by quantitating morphological indications of cell death, terminal dUTP nick-end labeling, and cleaved caspase 3 immunohistochemistry. Both UGN KO and GC-C KO mice were more susceptible than their WT littermates in this in vivo model of apoptotic injury. Furthermore, cGMP supplementation in both GC-C and UGN KO animals ameliorated radiation-induced apoptosis. Neither WT strain demonstrated significant alteration in apoptotic susceptibility as a result of cGMP supplementation before radiation injury. These in vivo findings demonstrate increased radiosensitivity of IECs in UGN and GC-C KO mice and a role for cGMP as a primary downstream mediator of GC-C activation in the protection of these IECs from radiation-induced apoptosis.

To investigate the expression of guanylyl cyclase-C (GC-C) and caudal type homeobox transcription factor 2 (CDX2) in human gastric mucosa at different stages and the significance thereof.An Immunofluorescence method was used to detect the expression of GC-C and CDX-2 in 23 specimens of gastric carcinoma and matching noncancerous tissues. Western blotting was used to detect the protein expression of GC-C and CDX2 in the gastric carcinoma tissues and matching noncancerous tissues too.The GC-C and CDX2 expression rates were 39.1% and 39.1% respectively in the intestinal metaplasia specimens, 55.6% and 55.6% respectively in the dysplasia specimens, and 56.7 % and 60.0% in the gastric carcinoma specimens, all significantly higher than those in the normal mucosa specimens (all P = 0.000) without significant differences in the expression of GC-C and CDX-2 among the 3 pathological groups. The GC-C and CDX-2 expression was positively correlated with Lauren classification, The expression levels of GC-C and CDX-2 were significantly higher in the intestinal-type than in the diffuse-type gastric carcinoma (P0.05). The GC-C expression was positively correlated with the expression of CDX-2 in intestinal metaplasia and gastric carcinoma.Ectopic expression of GC-C and CDX2 in human gastric mucosa may play an important role in the carcinogenesis of intestinal-type gastric carcinoma. Detection of GC-C and CDX2 helps diagnose gastric carcinoma and precursor lesions.

Mapping of the forces on biomolecules in cell membranes has spurred the development of effective labels, e.g. organic fluorophores and nanoparticles, to track trajectories of single biomolecules. Standard methods use particular statistics, namely the mean square displacement, to analyze the underlying dynamics. Here, we introduce general inference methods to fully exploit information in the experimental trajectories, providing sharp estimates of the forces and the diffusion coefficients in membrane microdomains. Rapid and reliable convergence of the inference scheme is demonstrated on trajectories generated numerically. The method is then applied to infer forces and potentials acting on the receptor of the $\epsilon$-toxin labeled by lanthanide-ion nanoparticles. Our scheme is applicable to any labeled biomolecule and results show show its general relevance for membrane compartmentation., Comment: 6 pages, 4 figures

Somatostatin analog SMS 201-995 causes a dose-related suppression of the release and/or action of several gastrointestinal hormones and impairs several anterior pituitary functions. Some patients with illness involving abnormal hormonal activity have responded to treatment with SMS 201-995, including resolution of severe secretory diarrhea. This study examined SMS 201-995 inhibition of Escherichia coli heat-stable enterotoxin STa (STa) effects and effects of the analog in the rabbit RITARD model with enterotoxigenic Escherichia coli. SMS 201-995 did not alter STa binding to its receptor on piglet brush border membranes. The analog, at concentrations of 0.1 micrograms/ml (0.1 microM) and 1 microgram/ml (1 microM) did not significantly alter STa activation of intestinal epithelial cell particulate guanylate cyclase. At maximal dosing the analog significantly reduced intestinal fluid secretion in suckling mice that was induced by either 8-bromo cyclic GMP or STa. In piglets, the analog reduced by 37-44% the amount of diarrhea induced by STa. However, even with maximal dosing, the piglets still had significant diarrhea, although of a lesser amount. In the rabbit RITARD model the drug failed to alter the severe diarrheal response seen when dosing the animals with enterotoxigenic Escherichia coli. Overall, SMS 201-995 had a significant but incomplete effect in reducing the STa effects seen in the various assays. Additionally, in the RITARD model the analog did not alter the clinical responses to various enterotoxigenic bacteria. SMS 201-995 should be useful as a probe into the mechanisms involved in intestinal fluid secretion, but a clinical role in enterotoxigenic gastrointestinal disease was not supported by this study.

Infection with Shiga toxin- and Shiga-like toxin-producing strains of Shigella dysenteriae and Escherichia coli, respectively, can progress to the hemolytic-uremic syndrome. It has been hypothesized that circulating Shiga toxin, Shiga-like toxins, and endotoxins may contribute to the disease by directly damaging glomerular endothelial cells. The effects of these toxins on HeLa, Vero, and human vascular endothelial cells (EC) were examined. Confluent EC were sensitive to Shiga toxin but were at least 10(6)-fold less sensitive to the toxins than were Vero cells. Shiga toxin was the predominant cytotoxic factor. Lipopolysaccharides were not cytotoxic and did not augment Shiga toxin-mediated toxicity. Lower doses of Shiga toxin caused cytotoxicity when coincubated with tumor necrosis factor. The relative resistance of EC to Shiga toxin and Shiga-like toxins may be due to reduced toxin binding, as low levels of globotriaosylceramide (Gb3), the toxin-specific receptor, were found in EC membranes.

Analogs of a heat-stable enterotoxin (ST) that have a CH 2 -S linkage instead of an S-S linkage in the molecule were synthesized by conventional methods. The synthetic peptides showed toxicity, assayed as induction of fluid secretion in suckling mice, although their toxicities were hundredth that of native ST. This finding implies that ST is not recognized by its receptor protein through an exchange reaction between its disulfide linkages and thiol-groups of its receptor proteins(s), but through hydrophobic or electrostatic interactions.

The staphylococcal enterotoxins (SE) bind to major histocompatibility complex (MHC) class II molecules on target cells and activate T cells expressing particular T cell receptor V beta sequences. In this report we demonstrate that SE bind to the MHC class II- SW620, Colo320DM and WiDr human colon carcinoma cell lines and direct cytotoxic T lymphocytes (CTL) to mediate strong target cell killing. Flow cytometry analysis, immunoprecipitation and Northern blotting experiments failed to demonstrate any surface expression of HLA-DR, HLA-DP and HLA-DQ isotypes on the SW620 colon carcinoma cell line, whereas abundant expression of these isotypes was seen on Raji cells, SEB and SEC1 were efficiently presented at picomolar concentration by the MHC class II- colon carcinoma cells and MHC class II+ Raji cells, whereas SEA and SED were preferentially presented on the MHC class II+ Raji cells. An anti-HLA-DR monoclonal antibody inhibited SEB-induced CTL targeting to Raji, but did not influence the killing of SW620 cells. Our data suggests the existence of functionally active SE-binding structures on human colon carcinoma cells which are distinct from the conventional MHC class II molecules. The possibility that these putative new SE receptors play a role in the enterotoxin action of SE must be considered.

The intestinal guanylyl cyclase-C (GC-C) was originally identified as an Escherichia coli heat-stable enterotoxin (STa) receptor. STa stimulates GC-C to much higher activity than the endogenous ligands guanylin and uroguanylin, causing severe diarrhea. To investigate the interactions of the endogenous and bacterial ligands with GC-C, we designed and characterized a soluble and properly folded fragment of the extracellular ligand-binding domain of GC-C. The membrane-bound guanylyl cyclases exhibit a single transmembrane spanning helix and a globularly folded extracellular ligand-binding domain that comprises about 410 of 1050 residues. Based on the crystal structure of the dimerized-binding domain of the guanylyl cyclase-coupled atrial natriuretic peptide receptor and a secondary structure-guided sequence alignment, we generated a model of the extracellular domain of GC-C comprised of two subdomains. Mapping of mutational and cross-link data onto this structural model restricts the ligand-binding region to the membrane proximal subdomain. We thus designed miniGC-C, a 197 amino acid fragment that mimics the ligand-binding membrane proximal subdomain. Cloning, expression and spectroscopic studies reveal miniGC-C to be a soluble and properly folded protein with a distinct secondary and tertiary structure. MiniGC-C binds STa with nanomolar affinity.

Previous studies have established a stimulatory effect of natriuretic peptides (NP) on testosterone production in mouse Leydig cells as intense as that of LH. Chronic administration of ANP in mice, on the other side, reduced testosterone levels. So, the understanding of the role of ANP on testicular steroidogenesis has been impaired by discrepant findings. The aim of the present study was to clarify the physiological role of ANP in the rat testis steroidogenesis using a model that preserves the interactions between testis cells and a medium devoid of any circulating factors that could interfere with testosterone production. First, ANP was immunolocalized in the interstitial compartment of the rat testis, mainly in Leydig cells. We also determined the presence of ANP and both GC-A (guanylyl cyclase A) and C receptors by real-time PCR in testis. Perfusion in vitro of testis with ANP (1 and 3x10(-7)M) stimulated testosterone production in a time- and dose-dependent manner. On the other side, testosterone secretion induced by LH was blunted by ANP. Similar effect was obtained using the specific C receptor ligand, cANF, indicating the involvement of C receptor in such response. In conclusion, ANP stimulated testosterone production in the rat testis perfused in vitro but decreased testosterone production LH-induced, effect that seems to involve C receptor. To this extent, our results suggest the existence of a local and complex peptidergic system in the rat testis, involving ANP and its receptors that could importantly modulate the androgen biosynthesis.

Gastrointestinal cancers remain a significant cause of morbidity and mortality. While increasing therapeutic options have improved outcomes for many patients, they have also complicated treatment decision-making. Unfortunately, most patients with advanced gastrointestinal malignancies die from their disease. Prognostic and predictive markers could improve treatment significantly by identifying patients who may or may not require a given therapy, and determining those most likely to benefit from a therapy. Candidates for such markers include blood antigens and circulating tumor cells, tumor enzyme and gene expression, and pharmacodynamic endpoints. In this review, we summarize reported and ongoing research to define and validate prognostic and predictive markers in gastrointestinal malignancies, with an emphasis on colorectal cancer and brief overview of pancreatic and neuroendocrine tumors.

In the North American opossum, heat-stable specific binding sites for E. coli enterotoxin are observed (i) in epithelial cells lining the small intestine, colon, gall bladder, cystic duct, common bile duct and trachea, and (ii) in epithelial cells forming the duodenal (Brunner's) glands, liver, kidneys (metanephros, mesonephros) and testis, as demonstrated by autoradiography. Enterotoxin-specific binding sites in the intestinal tract are only found in intestinal epithelial cells with the highest concentration in the microvillus border. Enterotoxin-specific binding sites also occur in epithelial cells comprising the secretory tubules and ducts of the duodenal glands. In the kidneys (metanephros and mesonephros), enterotoxin-specific binding sites are confirmed primarily to the proximal tubules, whereas in the testis they are localized in seminiferous tubules. In the liver, enterotoxin-specific binding sites are confined primarily to hepatocytes. E. coli enterotoxin caused a 7-fold increase of cGMP in the liver and a 30-fold increase in the duodenal glands. The liver responded in about half of the animals studied, whereas the duodenal glands gave a consistent response in each case. Likewise, the duodenal glands consistently showed strong labelling for 125I-enterotoxin, whereas receptor labelling of hepatocytes was inconsistent in nearly half the incubations and corresponds to the observed cGMP measurements.

The receptor for heat-stable enterotoxins (ST) produced by Escherichia coli and related organisms is located in the brush border region of intestinal villus cells. Heterobifunctional and homobifunctional crosslinkers were used to covalently couple 125I-ST to rat intestinal cell brush border membrane proteins. Experimental conditions during ligand binding and subsequent crosslinking significantly influence the efficiency of crosslinking, and the number of peptides specifically crosslinked to the 125I-ST. Multiple proteins efficiently coupled to 125I-ST with agents that can couple through the ST amino terminus. The crosslinker 1-ethyl-3-(dimethylaminopropyl)carbodiimide (EDC), which can react with the carboxy terminus of the ST, covalently crosslinked 125I-ST to a single protein with an apparent Mr of 125,000-130,000, larger than the proteins identified using longer crosslinkers. Each of the proteins identified by crosslinking migrate with the same retention time on gel filtration after solubilization, with an approximate molecular size of 150,000-200,000.

The heat-stable enterotoxin of Escherichia coli (STa) is a potent stimulant of intestinal chloride and bicarbonate secretion. Guanylyl cyclase C (GC-C) has been shown to be the primary receptor involved in mediating this response. However, numerous studies have suggested the existence of an alternative STa-binding receptor. The aims of this study were to determine whether a non-GC-C receptor exists for STa and what is the functional relevance of this for intestinal bicarbonate secretion in mice. (125)I-STa-binding experiments were performed with intestinal mucosae from GC-C knockout (KO) and wild type (WT) mice. Subsequently, the functional relevance of an alternative STa-binding receptor was explored by examining STa-, uroguanylin-, and guanylin-stimulated duodenal bicarbonate secretion (DBS) in GC-C KO mice in vitro and in vivo. Significant (125)I-STa-binding occurred in the proximal small intestines of GC-C KO and WT mice. Analysis of binding coefficients and pH dependence showed that (125)I-STa-binding in GC-C KO mice involved a receptor distinct from that of WT mice. Functionally, STa, uroguanylin, and guanylin all stimulated a significant increase in DBS in GC-C KO mice. Uroguanylin- and guanylin-stimulated DBS were significantly inhibited by glibenclamide, but not by 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS). However, STa-stimulated DBS was unaffected by glibenclamide but inhibited by DIDS. Taken together, our results suggest that alternative, non-GC-C, receptors likely exist for STa, uroguanylin, and guanylin in the intestines of mice. While uroguanylin- and guanylin-stimulated DBS are cystic fibrosis transmembrane conductance regulator (CFTR) dependent, STa-stimulated DBS is CFTR independent. Further understanding of this alternative receptor and its signaling pathway may provide important insights into rectification of intestinal bicarbonate secretion in cystic fibrosis.

Agonists of guanylyl-C receptor, such as guanylin/uroguanylin, are correlated not only with the intestinal cell epithelial physiology but also with the colorectal cancer tumorigenesis. Activation of the second intracellular messenger cyclic guanosine monophosphate by guanylyl cyclase-C receptor results in a complex intracellular signalling cascade involving the phosphodiesterase, the ion channels and the protein kinase. After an analytical review of relevant new knowledge, new diagnostic and therapeutic approaches for colorectal cancer are discussed.

Background & 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 (ApcMin/+) or exposed to the carcinogen azoxymethane. Markers of DNA damage, loss of Apc heterozygosity, and β-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 ApcMin/+ 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.

The elaboration of heat stable enterotoxin (STa) is an important step in the pathogenesis of enterotoxigenic Escherichia coli (ETEC), which causes severe diarrhea in newborn animals. In this study, the distribution of the STa-specific receptors on enterocytes and brush border membrane vesicles (BBMVs) prepared from the anterior jejunum, posterior jejunum, ileum and colon of newborn kids was investigated. The density of STa-receptors on enterocytes and BBMVs was higher in the posterior jejunum than that in other segments of the kids' intestines. Additionally, the affinity of the posterior jejunum STa-receptors was higher than the affinity of receptors present on the epithelium of other intestinal segments. Our findings suggest that the posterior jejunum is a major target for STa within the intestinal tract of newborn kids.

Purpose: Guanylyl cyclase C (GCC), a receptor for bacterial diarrheagenic enterotoxins, may be a prognostic and predictive marker to detect occult micrometastases in patients undergoing staging for colorectal cancer. However, quantification of GCC expression in tissues by the quantitative reverse transcription-PCR (qRT-PCR) has not undergone analytic and clinicopathologic validation. Experimental Design: A technique to quantify GCC mRNA in tissues employing RT-PCR was developed and validated employing external calibration standards of RNA complementary to GCC. Results: GCC qRT-PCR exhibited reaction efficiencies >92%, coefficients of variations 6 orders of magnitude, and a limit of quantification of >25 copies of GCC cRNA. This assay confirmed that GCC mRNA was overexpressed by colorectal tumors from 41 patients, which correlated with increased GCC protein quantified by immunohistochemistry. Analyses obtained with 164 lymph nodes from patients free of cancer and 15 nodes harboring metastases established a threshold for metastatic disease of ∼200 GCC mRNA copies/μg total RNA, with a sensitivity of 93% and specificity of 97%. GCC mRNA above that threshold was detected in 76 of 367 (∼21%) nodes free of disease by histopathology from 6 of 23 (26%) patients, suggesting the presence of occult micrometastases. Conclusions: Quantifying GCC mRNA in tissues by RT-PCR employing external calibration standards is analytically robust and reproducible, with high clinicopathologic sensitivity and specificity. This validated assay is being applied to ∼10,000 lymph nodes in a prospective trial to define the sensitivity of GCC qRT-PCR for staging patients with colorectal cancer.

Receptor guanylyl cyclases respond to ligand stimulation by increasing intracellular cGMP, thereby initiating a variety of cell-signaling pathways. Furthermore, these proteins are differentially localized at the apical and basolateral membranes of epithelial cells. We have identified a region of 11 amino acids in the cytosolic COOH terminus of guanylyl cyclase C (GCC) required for normal apical localization in Madin-Darby canine kidney (MDCK) cells. These amino acids share no significant sequence homology with previously identified cytosolic apical sorting determinants. However, these amino acids are highly conserved and are sufficient to confer apical polarity to the interleukin-2 receptor alpha-chain (Tac). Additionally, we find two molecular weight species of GCC in lysates prepared from MDCK cells over-expressing GCC but observe only the fully mature species on the cell surface. Using pulse-chase analysis in polarized MDCK cells, we followed the generation of this mature species over time finding it to be detectable only at the apical cell surface. These data support the hypothesis that selective apical sorting can be determined using short, cytosolic amino acid motifs and argue for the existence of apical sorting machinery comparable with the machinery identified for basolateral protein traffic.

Colorectal cancer is the third most common malignancy and the second most common cause of cancer-related mortality worldwide. While surgery remains the mainstay of therapy, approximately 50% of patients who undergo resection develop parenchymal metastatic disease. Unfortunately, current therapeutic regimens offer little improvement to the survival of patients with parenchymal metastases in the liver and lung. In that context, there is a significant unrealized opportunity at the intersection of engineering and biology for the development of novel targeted therapeutic approaches to colorectal cancer metastases. This opportunity exploits the discovery that an intestinal receptor, guanylyl cyclase C, which mediates diarrhea induced by bacterial heat-stable enterotoxins (STs), is over-expressed by metastatic colorectal tumors only. Moreover, it leverages recent advances in the fabrication of metal nanoshells with defined thicknesses absorb near-infrared (NIR) light, resulting in resonance and transfer of thermal energies of more than 40°C. Thus, the conjugation of ST to gold nanoshells, which can undergo resonance excitation by NIR light and emit heat, represents a previously unrecognized approach for the targeted therapy of parenchymal colorectal cancer metastases, specifically to the liver and lung. This article discusses the potential of ST-targeted nanoshells for NIR thermal ablation of metastatic colorectal tumors and highlights the significant challenges and solutions linked to the translation of this emerging technology to patient care.

We have previously shown that the partial disruption of the gene for atrial natriuretic peptide (ANP) results in a salt-sensitive phenotype. The present study examined the possibility that alterations in either the ANP natriuretic pathway or endothelin (ET) system in the kidney of the salt-challenged ANP +/− mouse was responsible for its salt-sensitive phenotype. Plasma ANP levels and renal cGMP activity were increased in response to a salt load in both ANP +/+ and +/− mice. However, the mRNA expression of proANP was found to be increased only in the ANP +/− kidney along with its guanylyl cyclase-linked receptor, NPRA; the upregulation of NPRA mRNA was limited to the renal medulla. This suggests that the renal ANP pathway remains capable of responding to a salt load in the ANP +/− animal, but may be compensating for other dysfunctional pathways. We also report a significant increase in renal ET-1 mRNA and ET A receptor protein expression in medulla and cortex of the salt-treated, ANP +/− mouse, but not its wild-type counterpart. In fact, ET A expression decreased in the renal cortex of the ANP +/+ salt-treated animal. The ET B receptor expression was not affected by diet in either genotype. We hypothesize that the salt-sensitive hypertension in the ANP +/− mouse is exacerbated, and possibly driven by the vasoconstrictive effects resulting from an upregulated ET-1/ET A pathway.

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.