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Guanylyl cyclase C (GCC), the receptor for diarrheagenic bacterial heat-stable enterotoxins (STs), inhibits colorectal cancer cell proliferation by co-opting Ca(2+) as the intracellular messenger. Similarly, extracellular Ca(2+) (Ca(2+)(o)) opposes proliferation and induces terminal differentiation in intestinal epithelial cells. In that context, human colon cancer cells develop a phenotype characterized by insensitivity to cytostasis imposed by Ca(2+)(o). Here, preconditioning with ST, mediated by GCC signaling through cyclic nucleotide-gated channels, restored Ca(2+)(o)-dependent cytostasis, reflecting posttranscriptional regulation of calcium-sensing receptors (CaRs). ST-induced GCC signaling deployed CaRs to the surface of human colon cancer cells, whereas elimination of GCC signaling in mice nearly abolished CaR expression in enterocytes. Moreover, ST-induced Ca(2+)(o)-dependent cytostasis was abrogated by CaR-specific antisense oligonucleotides. Importantly, following ST preconditioning, newly expressed CaRs at the cell surface represented tumor cell receptor targets for antiproliferative signaling by CaR agonists. Since expression of the endogenous paracrine hormones for GCC is uniformly lost early in carcinogenesis, these observations offer a mechanistic explanation for the Ca(2+)(o)-resistant phenotype of colon cancer cells. Restoration of antitumorigenic CaR signaling by GCC ligand replacement therapy represents a previously unrecognized paradigm for the prevention and treatment of human colorectal cancer employing dietary Ca(2+) supplementation.

Guanylyl cyclase C and accumulation of cGMP induced by bacterial heat-stable enterotoxins (STs) promote colon cancer cell cytostasis, serving as a tumor suppressor in intestine. Conversely, capacitative calcium entry through store-operated calcium channels (SOCs) is a key signaling mechanism that promotes colon cancer cell proliferation. The present study revealed that proliferative signaling by capacitative calcium entry through SOCs opposes and is reciprocally coupled to cytostasis mediated by guanylyl cyclase C in T84 human colon carcinoma cells. Elimination of capacitative calcium entry employing 2-aminoethoxydiphenylborate (2-APB), a selective inhibitor of SOCs, potentiated cytostasis induced by ST. Opposition of ST-induced cytostasis by capacitative calcium entry reflects reciprocal inhibition of guanylyl cyclase C signaling. Calcium entry through SOCs induced by the calcium-ATPase inhibitor thapsigargin or the receptor agonists UTP or carbachol inhibited guanylyl cyclase C-dependent cGMP accumulation. This effect was mimicked by the calcium ionophore ionomycin and blocked by 2-APB and intracellular 1,2-bis(o-amino-5,5'-dibromophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM), a chelator of calcium. Moreover, regulation by capacitative calcium entry reflected ligand-dependent sensitization of guanylyl cyclase C to inhibition by that cation. Although basal catalytic activity was refractory, ST-stimulated guanylyl cyclase C was inhibited by calcium, which antagonized binding of magnesium to allosteric sites required for receptor-effector coupling. These observations demonstrate that reciprocal regulation of guanylyl cyclase C signaling by capacitative calcium entry through SOCs represents one limb of a coordinated mechanism balancing colon cancer cell proliferation and cytostasis. They suggest that combining guanylyl cyclase C agonists and SOC inhibitors offers a novel paradigm for cGMP-directed therapy and prevention for colorectal tumors.

Guanylyl cyclase C (GCC) through cyclic GMP (cGMP) inhibits colorectal cancer cell proliferation, serving as a tumor suppressor in intestine. Conversely, Ca2+ entry through calcium release-activated calcium (Icrac) channels promotes colorectal cancer growth. The present study explored the functional relationship between mitogenic signaling by Ca2+ entry through Icrac channels and antiproliferative signaling by GCC in T84 human colon carcinoma cells. The E. coli heat-stable enterotoxin ST, a specific GCC ligand, induced intracellular accumulation of cGMP, Ca2+ entry through CNG channels and inhibition of human colorectal cancer cell proliferation. In contrast, influx of extracellular Ca2+ through Icrac channels, induced by thapsigargin, UTP or carbachol, inhibited GCC-dependent cGMP accumulation. Chelating intracellular Ca2+, removing extracellular Ca2+, or specifically blocking Icrac channels with the antagonist 2-APB reversed this inhibition. Moreover, 2-APB blocked Ca2+-dependent cell proliferation and amplified the anti-proliferative effects of ST. These data suggest that an imbalance of pro- and anti-mitogenic signals mediated by Ca2+ may contribute to mechanisms underlying colorectal carcinogenesis. In addition, they suggest that the combination of GCC agonists and Icrac channel inhibitors represents a novel paradigm for cGMP-directed therapy for colorectal tumors. Clinical Pharmacology & Therapeutics (2004) 75, P48–P48; doi: 10.1016/j.clpt.2003.11.180