The endocannabinoid anandamide inhibits cholangiocarcinoma growth via activation of the noncanonical Wnt signaling pathwaySharon DeMorrow, Heather Francis, Eugenio Gaudio, Julie Venter, Antonio Franchitto, Shelley Kopriva, Paolo Onori, Romina Mancinelli, Gabriel Frampton, Monique Coufal, Brett Mitchell, Bradley Vaculin, and Gianfranco Alpini
Am J Physiol Gastrointest Liver Physiol. 2008 December
Cholangiocarcinomas are cancers that have poor prognosis and limited treatment options. The noncanonical Wnt pathway is mediated predominantly by Wnt 5a, which activates a Ca2+-dependent pathway involving protein kinase C, or a Ca2+-independent pathway involving the orphan receptor Ror2 and subsequent activation of Jun NH2-terminal kinase (JNK). This pathway is associated with growth-suppressing effects in numerous cell types. We have shown that anandamide decreases cholangiocarcinoma growth in vitro. Therefore, we determined the effects of anandamide on cholangiocarcinoma tumor growth in vivo using a xenograft model and evaluated the effects of anandamide on the noncanonical Wnt signaling pathways. Chronic administration of anandamide decreased tumor growth and was associated with increased Wnt 5a expression in vitro and in vivo. Treatment of cholangiocarcinoma cells with recombinant Wnt 5a decreased cell proliferation in vitro. Neither anandamide nor Wnt 5a affected intracellular calcium release, but both increased the JNK phosphorylation. Stable knockdown of Wnt 5a or Ror2 expression in cholangiocarcinoma cells abolished the effects of anandamide on cell proliferation and JNK activation. Modulation of the endocannabinoid system may be important in cholangiocarcinoma treatment. The antiproliferative actions of the noncanonical Wnt signaling pathway warrants further investigation to dissect the mechanism by which this may occur.
Cholangiocarcinomas are devastating cancers of intrahepatic and extrahepatic origin that are increasing in both their worldwide incidence and mortality rate (2, 40). The challenges posed by these often lethal biliary tract cancers are daunting, with conventional treatment options being limited and the only hope for long-term survival being that of complete surgical resection of the tumor (2, 40). Conventional chemotherapy and radiation therapy are not effective in prolonging long-term survival (2); therefore, it is important to understand the cellular mechanisms of cholangiocarcinoma cell growth with a view to develop novel chemopreventive strategies.
Marijuana and its derivatives have been used in medicine for many centuries, and, presently, there is an emerging renaissance in the study of the therapeutic effects of cannabinoids. Ongoing research is determining that regulation of the endocannabinoid system may be effective in the treatment of pain (6, 29), glaucoma (48), and neurodegenerative disorders such as Parkinson's disease (33) and multiple sclerosis (3). In addition, cannabinoids might be effective antitumoral agents because of their ability to inhibit the growth of various types of cancer cell lines in culture (9, 35–37) and in laboratory animals (16). Indeed, we have recently demonstrated that the endocannabinoid anandamide (AEA) has antiproliferative effects on cholangiocarcinoma cell lines in vitro via a cannabinoid receptor-independent pathway involving the stabilization of lipid raft-membrane structures and the recruitment of death-receptor complexes into the lipid rafts (11). Conversely, we have also shown that disruption of lipid raft integrity by the endocannabinoid 2-arachidonoyl glycerol leads to increased cholangiocarcinoma cell proliferation (11). The downstream mechanisms of the antiproliferative effects of AEA on cholangiocarcinoma are unknown.
Wnt signaling proteins are known to exert an effect on a wide variety of developmental and pathological processes (42). They can be divided further into two subclasses: the canonical pathway (Wnt/β-catenin pathway) and the noncanonical pathway (Wnt/Ca2+ or Wnt/JNK pathway) (21). The canonical Wnt signaling pathway involves the inhibition of glycogen synthase kinase-3β and subsequent accumulation and nuclear translocation of β-catenin into the nucleus (30). This pathway is thought to be overactive in various cancers (15). However, the noncanonical pathway, including Wnt 5a, is more complex. Activation of this pathway is thought to trigger the intracytoplasmic release of Ca2+ and the activation of subsequent Ca2+-related signaling (41). Alternatively, Wnt 5a can interact with the receptor tyrosine kinase orphan receptor Ror2 to activate a JNK-mediated pathway (38). The consequences of noncanonical Wnt signaling activation on cell proliferation are unclear. On the one hand, Wnt 5a has tumor-suppressive properties in acute lymphoblastic leukemia (34) and thyroid carcinoma (24). Conversely, Wnt 5a has also been shown to increase endothelial cell proliferation and migration (8).
To date, the effects of the noncanonical Wnt signaling pathway on cholangiocarcinoma growth have not been studied; however, Ca2+ signaling has been shown to have opposing effects on cholangiocyte cell growth (17, 18). Vascular endothelial growth factor (VEGF), for example, stimulates the growth of normal cholangiocytes by activation of the inositol triphosphate (IP3)/Ca2+ signaling pathway with subsequent phosphorylation of Src and ERK1/2 (17), and the hormone gastrin has been shown to inhibit cholangiocyte hyperplasia by activation of the IP3/Ca2+/PKC signaling pathway (18). A role of calcium signaling in the control of neoplastic growth has also been established (1, 12, 22). Increased cytosolic Ca2+ may be responsible for the decrease in cholangiocarcinoma growth observed after administration of tauroursodeoxycholate (1), gastrin (22), and γ-aminobutyric acid (12). The effects of Ror2 activation on cholangiocyte and cholangiocarcinoma cell growth, however, are unknown.
The aims of this study were 1) to demonstrate the antiproliferative effects of anandamide in an in vivo xenograft model of cholangiocarcinoma, 2) to determine the effects of anandamide on the noncanonical Wnt signaling pathway and to identify downstream effectors of this pathway, and 3) to evaluate the role of Wnt signaling on cholangiocarcinoma cell growth.
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