Prostaglandins (PGs), bioactive lipid molecules produced by cyclooxygenase enzymes (COX-1 and COX-2), have diverse biological activities, including growth-promoting actions on gastrointestinal mucosa. They are also implicated in the growth of colonic polyps and cancers. However, the precise mechanisms of these trophic actions of PGs remain unclear. As activation of the epidermal growth factor receptor (EGFR) triggers mitogenic signaling in gastrointestinal mucosa, and its expression is also upregulated in colonic cancers and most neoplasms, we investigated whether PGs transactivate EGFR. Here we provide evidence that prostaglandin E2 (PGE2) rapidly phosphorylates EGFR and triggers the extracellular signal-regulated kinase 2 (ERK2)--mitogenic signaling pathway in normal gastric epithelial (RGM1) and colon cancer (Caco-2, LoVo and HT-29) cell lines. Inactivation of EGFR kinase with selective inhibitors significantly reduces PGE2-induced ERK2 activation, c-fos mRNA expression and cell proliferation. Inhibition of matrix metalloproteinases (MMPs), transforming growth factor-alpha (TGF-alpha) or c-Src blocked PGE2-mediated EGFR transactivation and downstream signaling indicating that PGE2-induced EGFR transactivation involves signaling transduced via TGF-alpha, an EGFR ligand, likely released by c-Src-activated MMP(s). Our findings that PGE2 transactivates EGFR reveal a previously unknown mechanism by which PGE2 mediates trophic actions resulting in gastric and intestinal hypertrophy as well as growth of colonic polyps and cancers.

Although fibroblast growth factor 19 (FGF19) can promote liver carcinogenesis in mice its involvement in human cancer is not well characterized. Here we report that FGF19 and its cognate receptor FGF receptor 4 (FGFR4) are coexpressed in primary human liver, lung and colon tumors and in a subset of human colon cancer cell lines. To test the importance of FGF19 for tumor growth, we developed an anti-FGF19 monoclonal antibody that selectively blocks the interaction of FGF19 with FGFR4. This antibody abolished FGF19-mediated activity in vitro and inhibited growth of colon tumor xenografts in vivo and effectively prevented hepatocellular carcinomas in FGF19 transgenic mice. The efficacy of the antibody in these models was linked to inhibition of FGF19-dependent activation of FGFR4, FRS2, ERK and beta-catenin. These findings suggest that the inactivation of FGF19 could be beneficial for the treatment of colon cancer, liver cancer and other malignancies involving interaction of FGF19 and FGFR4.

We have recently shown that Neuropeptide S (NPS) can promote arousal and induce anxiolytic-like effects after central administration in rodents. Another study reported a number of natural polymorphisms in the human NPS receptor gene. Some of these polymorphisms were associated with increased risk of asthma and possibly other forms of atopic diseases, but the physiological consequences of the mutations remain unclear. One of the polymorphisms produces an Asn-Ile exchange in the first extracellular loop of the receptor protein, and a C-terminal splice variant of the NPS receptor was found overexpressed in human asthmatic airway tissue. We sought to study the pharmacology of the human receptor variants in comparison with the murine receptor protein. Here, we report that the N107I polymorphism in the human NPS receptor results in a gain-offunction characterized by an increase in agonist potency without changing binding affinity in NPSR Ile 107 . In contrast, the C-terminal splice variant of the human NPS receptor shows a pharmacological profile similar to NPSR Asn 107 . The mouse NPS receptor, which also carries an Ile residue at position 107, displays an intermediate pharmacological profile. Structureactivity relationship studies show that the amino terminus of NPS is critical for receptor activation. The altered pharmacology of the Ile 107 isoform of the human NPS receptor implies a mechanism of enhanced NPS signaling that might have physiological significance for brain function as well as peripheral tissues that express NPS receptors.Neuropeptide S (NPS) is the endogenous ligand of an orphan G protein-coupled receptor (GPCR). The NPS receptor (NPSR) belongs to the subfamily of peptide GPCRs and is widely expressed in the brain, with highest levels found in hypothalamus, amygdala, endopiriform nucleus, cortex, subiculum, and nuclei of the thalamic midline. In contrast, the NPS precursor mRNA is found in only a few brain structures (Xu et al., 2004). Highest levels of NPS precursor expression were detected in a novel nucleus located in between the noradrenergic locus coeruleus and Barrington's nucleus in the pontine area of the rat brain stem. Other brain regions of high NPS precursor expression include the lateral parabrachial nucleus, sensory principle 5 nucleus, and a few scattered neurons in the amygdala and dorsomedial hypothalamic nucleus. In addition, we found high expression of NPS and NPSR mRNA in endocrine tissues, including thyroid, mammary, and salivary glands, but did not observe significant levels in rat lung tissue.Central administration of NPS promotes behavioral arousal and suppresses all stages of sleep in rodents. Furthermore, NPS was found to produce anxiolytic-like effects in a battery of four different tests that measure behavioral responses of rodents to novelty or stress. NPS was shown to induce transient increases of intracellular Ca 2ϩ , indicating that it might have excitatory effects at the cellular level (Xu et al., 2004).Recently, a number of polymorphisms in the human NPS rece...

Colorectal cancer is the second most frequent cancer in the Western world, often lethal when invasion and/or metastasis occur. In addition to hepatocyte growth factor (HGF), colon cancer invasion may be driven by prostaglandins, especially the E2 series (PGE2), generated by the cyclooxygenase-2 (Cox-2) enzyme. While concentration of PGE2 as well as expression of Cox-2, HGF receptor (c-Met-R), epidermal growth factor receptor (EGFR), and beta-catenin are all dramatically increased in colon cancers and implicated in their growth and invasion, the precise role of PGE2 in the latter process remains unclear. Here we provide evidence that PGE2 transactivates c-Met-R (contingent upon functional EGFR), increases tyrosine phosphorylation and nuclear accumulation of beta-catenin, and induces urokinase-type plasminogen activator receptor (uPAR) mRNA expression. This is accompanied by increased beta-catenin association with c-Met-R and enhanced colon cancer cell invasiveness. Inactivation of EGFR and c-Met-R significantly reduced PGE2-induced cancer cell invasiveness. Clinical relevance of these findings is confirmed by our immunohistochemical studies demonstrating that cancer cells in the invasive front overexpress Cox-2, c-Met-R, and beta-catenin. Our findings explain a functional relationship between prostaglandins, EGFR, and c-Met-R in colon cancer growth and invasion.

Colorectal cancer is often lethal when invasion and/or metastasis occur. Tumor progression to the metastatic phenotype is mainly dependent on tumor cell invasiveness. Secondary bile acids, particularly deoxycholic acid (DCA), are implicated in promoting colon cancer growth and progression. Whether DCA modulates beta-catenin and promotes colon cancer cell growth and invasiveness remains unknown. Because beta-catenin and its target genes urokinase-type plasminogen activator receptor (uPAR) and cyclin D1 are overexpressed in colon cancers, and are linked to cancer growth, invasion, and metastasis, we investigated whether DCA activates beta-catenin signaling and promotes colon cancer cell growth and invasiveness. Our results show that low concentrations of DCA (5 and 50 microM) significantly increase tyrosine phosphorylation of beta-catenin, induce urokinase-type plasminogen activator, uPAR, and cyclin D1 expression and enhance colon cancer cell proliferation and invasiveness. These events are associated with a substantial loss of E-cadherin binding to beta-catenin. Inhibition of beta-catenin with small interfering RNA significantly reduced DCA-induced uPAR and cyclin D1 expression. Blocking uPAR with a neutralizing antibody significantly suppressed DCA-induced colon cancer cell proliferation and invasiveness. These findings provide evidence for a novel mechanism underlying the oncogenic effects of secondary bile acids.

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Fibroblast growth factors (FGF) play important roles in development, angiogenesis, and cancer. FGF19 uniquely binds to FGF receptor 4 (FGFR4). Our previous study has shown that FGF19 transgenic tumors have an activated Wnt-pathway phenotype. Wnt signaling is implicated in initiating or promoting FGF signaling in various cell types and organs. In this study, we examined whether FGF19 or inhibition of FGF19 affects the B-catenin signaling pathway using human colon cancer cell lines (HCT116, Colo201). Our results show that FGF19 increases tyrosine phosphorylation of B-catenin and causes loss of B-catenin-E-cadherin binding. FGF19 increases p-GSK3B and active B-catenin levels and anti-FGF19 antibody (1A6) treatment abrogates this effect of FGF19. Anti-FGF19 antibody treatment increases S33/S37/T41 phosphorylation and ubiquitination of B-catenin. Ion-trap mass spectrometric analysis confirmed that 1A6 increases phosphorylation of B-catenin in the NH 2 terminus. Using HCT116-paired B-catenin knockout cells, we show that FGF19 induces TCF/LEF reporter activity in parental (WT/#45) and in WT/À but not in mutant (À/#45) cells, and that inhibition of endogenous FGF19 reduces this reporter activity, indicating that wild-type Bcatenin is accessible for modulation. FGFR4 knockdown using inducible short hairpin RNA significantly reduces the colonyforming ability in vitro and tumor growth in vivo. Although cleaved caspase-3 immunoreactivity remains unchanged, the number of ki67-positive nuclei is reduced in FGFR4 knockdown tumor xenograft tissues. Consistent with the reduced B-catenin activation, Taqman analyses show that FGF19/FGFR4 inhibition reduced B-catenin target gene (cyclin D1, CD44, c-jun, Cox-2, UPAR) expression. These findings highlight that FGF19/FGFR4 cross-talk with B-catenin and that pathway intervention reduces tumor growth. [Cancer Res 2008; 68(13):5086-95]

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