Guanylyl cyclase C suppresses intestinal tumorigenesis by restricting proliferation and maintaining genomic integrity

BACKGROUND AND AIMS: The most commonly lost gene products in colorectal carcinogenesis include guanylin and uroguanylin, endogenous ligands for guanylyl cyclase C (GCC). Beyond intestinal fluid balance, GCC mediates diarrhea induced by bacterial enterotoxins, and an inverse relationship exists between enterotoxigenic Escherichia coli infections producing the exogenous GCC ligand ST and colorectal cancer worldwide. However, the role of GCC in neoplasia remains obscure. METHODS: Intestinal tumorigenesis was examined in wild-type (Gcc(+/+)) and GCC-deficient (Gcc(-/-)) mice carrying mutations in Apc (Apc(Min/+)) or exposed to the carcinogen azoxymethane. Markers of DNA damage, loss of Apc heterozygosity, and beta-catenin mutations were used to assess genomic integrity. Hyperproliferation was explored using Ki67 and cell cycle markers. Apoptosis was quantified by transferase biotin-dUTP nick end labeling analysis. RESULTS: In colons of Apc(Min/+) mice, deletion of Gcc increased tumor incidence and multiplicity, reflecting uncoupling of loss of genomic integrity and compensatory apoptosis. Conversely, in the small intestine, elimination of Gcc increased tumorigenesis by enhancing proliferation without altering genomic integrity. Moreover, these distinct but mutually reinforcing mechanisms collaborate in azoxymethane-exposed mice, and deletion of Gcc increased tumor initiation and growth associated with hypermutation and hyperproliferation, respectively, in conjunction with attenuated apoptosis. CONCLUSIONS: GCC suppresses tumor initiation and growth by maintaining genomic integrity and restricting proliferation. This previously unrecognized role of GCC in inhibiting tumorigenesis, together with the invariant disruption in guanylin and uroguanylin expression early in carcinogenesis, and the uniform over-expression of GCC by tumors, underscores the potential of oral administration of GCC ligands for targeted prevention and therapy of colorectal cancer.     

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

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.     

Pyridopyrimidine derivatives as inhibitors of cyclic nucleotide synthesis: Application for treatment of diarrhea

Acute secretory diarrhea induced by infection with enterotoxigenic strains of Escherichia coli involves binding of stable toxin (STa) to its receptor on the intestinal brush border, guanylyl cyclase type C (GC-C). Intracellular cGMP is elevated, inducing increase in chloride efflux and subsequent accumulation of fluid in the intestinal lumen. We have screened a library of compounds and identified a pyridopyrimidine derivatives {5-(3-bromophenyl)-1,3-dimethyl-5,11-dihydro-1H-indeno[2',1':5,6]pyrido[2,3-d]pyrimidine-2,4,6-trione; BPIPP} as an inhibitor of GC-C that can suppress STa-stimulated cGMP accumulation by decreasing GC-C activation in intact T84 human colorectal carcinoma cells. BPIPP inhibited stimulation of guanylyl cyclases, including types A and B and soluble isoform in various cells. BPIPP suppressed stimulation of adenylyl cyclase and significantly decreased the activities of adenylyl cyclase toxin of Bordetella pertussis and edema toxin of Bacillus anthracis. The effects of BPIPP on cyclic nucleotide synthesis were observed only in intact cells. The mechanism of BPIPP-dependent inhibition appears to be complex and indirect, possibly associated with phospholipase C and tyrosine-specific phosphorylation. BPIPP inhibited chloride-ion transport stimulated by activation of guanylyl or adenylyl cyclases and suppressed STa-induced fluid accumulation in an in vivo rabbit intestinal loop model. Thus, BPIPP may be a promising lead compound for treatment of diarrhea and other diseases.     

An alternatively spliced rat mineralocorticoid receptor mRNA causing truncation of the steroid binding domain

We attempted to clone the putative 11-dehydrocorticosterone receptor by RT-PCR with two degenerate primers from highly homologous regions of the DNA and steroid binding domains of the receptor subfamily. In doing so, we have identified an alternatively spliced variant mRNA of the rat mineralocorticoid (MR) with a ten bp deletion in the C-terminal steroid binding domain. This deletion results in a truncated MR receptor of 807 amino acids in comparison to the wild type of 981 amino acids. The deletion variant was expressed in colon, kidney, heart, liver, aorta and brain tissues. The relative abundance of the deletion variant compared to the wild type MR was estimated to be 6% in rat kidney and 4% in hippocampus. This deletion was also detected in human kidney by RT-PCR. Site-directed mutagenesis was used to create the eukaryotic expression plasmid pCR3-rMRdel10 from the wild type for a transactivation assay using the luciferase reporter system in CV-1 cells. The deletion variant had the same baseline transactivation activity as the wild type MR, but did not respond to aldosterone or corticosterone stimulation. Co-transfection of MR with the deletion variant had no significant effect on transactivation activity of the MR, indicating that the deletion variant is unlikely to serve as a negative regulator of MR function.     

Ornithine decarboxylase gene is overexpressed in colorectal carcinoma

AIM: To investigate the ornithine decarboxylase (ODC) gene expression in colorectal carcinoma, ODC mRNA was assayed by RT-PCR and ODC protein was detected by a monoclonal antibody against fusion of human colon ODC prepared by hybridoma technology. METHODS: Total RNA was extracted from human colorectal cancer tissues and their normal counterpart tissues. ODC mRNA levels were examined by RT-PCR. ODC genes amplified from RT-PCR were cloned into a prokaryotic vector pQE-30. The expressed proteins were purified by chromatography. Anti-ODC mAb was prepared with classical hybridoma techniques and used to determine the ODC expression in colon cancer tissues by immunohistochemical and Western blotting assay. RESULTS: A cell line, which could steadily secrete anti-ODC mAb, was selected through subcloning four times. Western blotting reconfirmed the mAb and ELISA showed that its subtype was IgG2a. RT-PCR showed that the ODC mRNA level increased greatly in colon cancer tissues (P<0.01). Immunohistochemical staining showed that colorectal carcinoma cells expressed a significantly higher level of ODC than normal colorectal mucosa (98.6±1.03% vs5.26±5%,P<0.01). CONCLUSION: ODC gene overexpression is significantly related to human colorectal carcinoma. ODC gene expression may be a marker for the gene diagnosis and therapy of colorectal carcinoma.     

Escherichia coli heat-stable enterotoxin receptors

PURPOSE: Receptors for Escherichia coli heat-stable toxin (ST) are selectively expressed in membranes of intestinal mucosa cells and colon carcinoma cells in vitro,suggesting their use as a marker for colorectal tumors in vivo.The present studies examined the expression and function of ST receptors in normal human tissues and primary and metastatic colorectal tumors obtained from patients at surgery. METHODS: Surgical specimens were obtained as follows: from normal colon; from primary adenocarcinomas from all anatomic divisions of the colon and rectum; from gallbladder, kidney, liver, lung, lymph node, ovary, peritoneum, stomach; and from colon carcinomas metastatic to liver, lung, lymph node, ovary, and peritoneum. Membranes prepared from these specimens were assessed for the presence and functional characteristics of ST receptors. RESULTS: ST bound specifically to membranes from each division of normal colon and rectum and all primary and metastatic colorectal tumors examined. The affinity and density of ST receptors were similar in tumors of different grades and from various metastatic sites. ST-receptor interaction was coupled to activation of guanylyl cyclase in all normal samples of colon and rectum and all primary and metastatic colorectal tumors examined. In contrast, neither ST binding nor ST activation of guanylyl cyclase was detected in any extraintestinal tissues examined. CONCLUSIONS: Functional ST receptors are expressed in normal colonic tissue and primary and metastatic colorectal tumors but not by extraintestinal tissues in humans. Expression of ST receptors does not vary as a function of the metastatic site or grade of these tumors. Receptors expressed by colorectal tumors retain their characteristic function, with binding of ST coupled to activation of guanylyl cyclase. These studies support the suggestion that ST receptors represent a specific marker for human colorectal tumors that may have use as a target for directing diagnostics and therapeutics to these tumors in vivo.Supported, in part, by grants from the National Science Foundation (IBN-9205717), the National Institutes of Health (1 R55 DK43805), the Elsa U. Pardee Foundation, and Targeted Diagnostics and Therapeutics, Inc. Stephen L. Carrithers was the recipient of a National Institutes of Health Postdoctoral Fellowship (1 F32 CA63764-01).Poster presentation at the meeting of The American Society of Colon and Rectal Surgeons, Montreal, Quebec, Canada, May 7 to 12, 1995.     

Two membrane forms of guanylyl cyclase found in the eye.

The cDNAs for two membrane guanylyl cyclases, designated E (GC-E) and F (GC-F, were isolated from a rat eye cDNA library. Their deduced topographic structures correspond to known members of the guanylyl cyclase receptor family, containing an extracellular domain, a single membrane-spanning domain, a protein kinase-like domain, and a cyclase catalytic domain. GC-E was expressed in the eye and the pineal gland, whereas GC-F expression was confined to the eye. Overproduction of GC-E and GC-F in COS cells resulted in expression of guanylyl cyclase activity, but ligands known to activate other guanylyl cyclase receptors failed to stimulate enzyme activity. Thus, both GC-E and GC-F remain orphan receptors. Amino acid sequence similarity between GC-E and GC-F in the extracellular region and homology with a cyclase expressed in olfactory neurons and retGC, a rod outer-segment-specific cyclase, suggest that there is another subfamily of guanylyl cyclase receptors, possibly restricted to sensory tissues.     

Evaluation of heterodimeric guanylyl cyclase genes as candidates for human hypertension

OBJECTIVE: Both physiologic and pharmacological data have implicated the nitric oxide (NO) signaling cascade in the regulation of blood pressure in humans and its impairment in the pathogenesis of hypertension. In biological systems, the principal receptor for NO is NO-stimulated guanylyl cyclase. NO-stimulated guanylyl cyclases are obligate heterodimers (alpha/beta). The genes for guanylyl cyclase subunits alpha1, beta, and beta2 are likely candidates for causing hypertension in the Dahl rat as their expression is altered and their gene loci are closely linked to known quantitative trait loci for blood pressure in Dahl rat crosses. The objective of the current study was to test whether markers near guanylyl cyclase subunit genes were linked to hypertension in Caucasians. DESIGN: To test for linkage of genetic markers in or near the guanylyl cyclase genes to hypertension in Caucasians, a sample of 124 Utah hypertensive sib pairs was genotyped. RESULTS: Four highly polymorphic markers in or near the human guanylyl cyclase subunits homologous to the rat alpha1 (human chromosome 8), rat beta1 (human chromosome 4), and rat beta2 (human chromosome 13) genes showed no evidence of excess allele sharing in the set of hypertensive sibships. CONCLUSION: We conclude that the heterodimeric guanylyl cyclase subunit loci do not appear to be linked to hypertension in Caucasians.     

Critical roles of the guanylyl cyclase B receptor in endochondral ossification and development of female reproductive organs

Guanylyl cyclase B is the receptor for a small peptide (C-type natriuretic peptide) produced locally in many different tissues. To unravel the functions of the receptor, we generated mice lacking guanylyl cyclase B through gene targeting. Expression of the receptor mRNA in tissues such as bone and female reproductive organs was evident, and significant phenotypes associated with each of these tissues were apparent in null mice. A dramatic impairment of endochondral ossification and an attenuation of longitudinal vertebra or limb-bone growth were seen in null animals. C-type natriuretic peptide-dependent increases of guanylyl cyclase B activity, but not basal enzyme activity, appeared to be required for the progression of endochondral ossification. Female mice were infertile, but male mice were not. This result was due to the failure of the female reproductive tract to develop. Thus, the guanylyl cyclase B receptor is critical for the development of both bone and female reproductive organs.