Diarrhea or colorectal cancer: can bacterial toxins serve as a treatment for colon cancer?

C olorectal cancer exhibits a low incidence in under-developed countries even though it is the third most common neoplasm worldwide and the second most common in the U.S. (1, 2). This geographical imbalance suggests an environmental contribution to the resistance of endemic populations to intestinal neoplasia. Although the epidemiology of colon cancer remains poorly understood, there is clearly an unexplained inverse relationship between the incidence of colorectal cancer and enterotoxigenic Escherichia coli (ETEC) infections (3, 4). Drawing from these observations, Pitari et al. (5) introduced an interesting hypothesis that specific peptides (STa) elaborated from ETEC may prevent the hyperproliferative and neoplastic development of intestinal epithelial cells that are associated with initiation and progression of colorectal cancer. Although a direct causal relationship between STa-mediated infectious diarrhea and low cancer rates in under-developed countries has not been proven, the authors provide convincing evidence of the presence of a novel intracellular signaling pathway initiated by STa that prevents proliferation of colon cancer cells. Mechanistically, STa binds to guanylyl cyclase-C (GC-C) receptors specifically expressed in intestinal cells (6). Ligand binding to GC-C activates the intracellular synthesis of the second messenger, cGMP. STa hyperactivates this signaling receptor, causing large increases of intracellular cGMP ([cGMP]int) levels (7). Despite this strong negative selective pressure of STa-mediated infection and diarrhea, GC-C and its signaling pathway have been evolutionarily conserved in a wide variety of animal species, suggesting that it must play a role in an important aspect of intestinal physiology. In this way, STa peptides represent molecular mimicry wherein enterotoxigenic bacteria have evolved a strategy for its transmission that exploits normal intestinal physiology (8, 9). Indeed, STa peptides are structurally and functionally homologous to the endogenous peptides guanylin and uroguanylin, which mediate autocrine paracrine control of intestinal f luid and electrolyte homeostasis (9). Recently, a new role for GC-C has been implied: a tumor suppressor. In fact, GC-C and its ligands have been implicated in the regulation of the balance of proliferation and differentiation along the crypt-to-villus axis in the intestine (10, 11). Interestingly, expression of guanylin and uroguanylin is lost during colon cancer tumorigenesis (12–14). In support of these studies, targeted inactivation of the mouse guanylin gene results in increased colonic epithelial proliferation (15). As a result, subsequent loss of the initiation of GC-C signaling may represent one key mutational event underlying neoplastic transformation in the colon. However, intestinal GC-C and its downstream intracellular effector molecules are conserved in colorectal tumors (16, 17), thus providing a means of restoring the signaling cascade associated with the tumor suppressor phenotype. In this manner, Forte and coworkers demonstrated that oral administration of uroguanylin suppresses the formation and progression of adenomatous polyps in the Min mouse animal model of colorectal cancer (13, 18). Mechanisms by which the GC-C ligands repress proliferation and colorectal tumorigenesis are unknown. In a recent issue of PNAS, Pitari et al. (5) demonstrated the presence of a previously unrecognized STa GC-C-induced cGMP-dependent signaling pathway, through cyclic nucleotide-gated (CNG) channels and calcium, responsible for the antiproliferative action of bacterial enterotoxins on human colon carcinoma cells (5). Similar to results shown from previous studies (10), STamediated inhibition of DNA synthesis and cellular proliferation in colon cancer cells (17, 19) was GC-C-dependent because controls using colon cancer cells devoid of GC-C (i.e., SW480 cells; ref. 19) were without effect. Commensurate with the toxin-induced effect was a concomitant increase in [cGMP]int. In fact, 8-Br-cGMP, a cell-permeable, nonhydrolyzable cGMP analog mimicked the antiproliferative effects of STa in colon cancer cells. In addition, inhibitors of colonic cell-specific cGMP-dependent phosphodiesterases, which catabolize cGMP to its respective biologically inactive noncyclic nucleotide 5 -GMP, potentiate antiproliferation by STa in human colon carcinoma cells, presumably by increasing the accumulation of [cGMP]int. Pitari et al. (10) further demonstrated that this GC-C mediated inhibition of proliferation was associated with a reduced rate of DNA synthesis. Uroguanylin mimicked the antiproliferative and cytostatic effects of STa in colon cancer cells whereas an inactive-analog of STa was without effect, suggesting the specificity and requirement for the STa uroguanylin receptor, GC-C. How does cGMP suppress proliferation of colon cancer cells? To address this question, Pitari et al. examined the antiproliferative effects of intracellular effector molecules of cGMP by STa in T84 cells by using a pharmacologic approach. Selective inhibitors of protein kinase G (PKG) and protein kinase A (PKA), which are previously characterized downstream effectors of cGMP and STa-mediated intestinal secretion (20, 21), did not influence the inhibition of proliferation by STa. Inhibition of phosphodiesterase 3, a target for negative cross-talk with cGMP pathways and an activator of the PKA pathway (22), also had no effect on STa-induced inhibition of DNA synthesis and tumor cell growth. Thus, conventional downstream effectors of cGMP did not mediate the antiproliferative effects of STa on human colon carcinoma cells. cGMP can also exert its actions through direct activation of cyclic nucleotide-gated (CNG) channels and or inhibition of Na Ca2 exchange, leading to alterations in intracellular calcium ([Ca2 ]int) (21). In fact, apart from cGMP, STa induces intracellular signaling through other second messengers (23, 24). In this respect, calcium does play a pivotal role. It should be noted that most, if not all, of the studies showing rise of [Ca2 ]int by STa have been restricted to the enterocytes of animals (25, 26). On the other hand, reports in T84 colon cancer cells and colonic epithelia are inconsistent. Although the chloride secretory responses in colonic cells have been shown to be influenced by both STa and calcium, a synergistic elevation of [Ca2 ]int by STa has yet to be observed (27, 28). These observa-