- Short communication
- Open Access
Protein kinase A antagonist inhibits β-catenin nuclear translocation, c-Myc and COX-2 expression and tumor promotion in ApcMin/+ mice
© Brudvik et al; licensee BioMed Central Ltd. 2011
Received: 7 June 2011
Accepted: 15 December 2011
Published: 15 December 2011
The adenomatous polyposis coli (APC) protein is part of the destruction complex controlling proteosomal degradation of β-catenin and limiting its nuclear translocation, which is thought to play a gate-keeping role in colorectal cancer. The destruction complex is inhibited by Wnt-Frz and prostaglandin E2 (PGE2) - PI-3 kinase pathways. Recent reports show that PGE2-induced phosphorylation of β-catenin by protein kinase A (PKA) increases nuclear translocation indicating two mechanisms of action of PGE2 on β-catenin homeostasis.
Treatment of ApcMin/+ mice that spontaneously develop intestinal adenomas with a PKA antagonist (Rp-8-Br-cAMPS) selectively targeting only the latter pathway reduced tumor load, but not the number of adenomas. Immunohistochemical characterization of intestines from treated and control animals revealed that expression of β-catenin, β-catenin nuclear translocation and expression of the β-catenin target genes c-Myc and COX-2 were significantly down-regulated upon Rp-8-Br-cAMPS treatment. Parallel experiments in a human colon cancer cell line (HCT116) revealed that Rp-8-Br-cAMPS blocked PGE2-induced β-catenin phosphorylation and c-Myc upregulation.
Based on our findings we suggest that PGE2 act through PKA to promote β-catenin nuclear translocation and tumor development in ApcMin/+ mice in vivo, indicating that the direct regulatory effect of PKA on β-catenin nuclear translocation is operative in intestinal cancer.
The adenomatous polyposis coli (APC) gene is thought to play a gate-keeping role in the tumor formation and progression and is the most commonly mutated gene in all colorectal cancers. In humans, APC mutations can be acquired (spontaneous CRC) or inherited as in the autosomal, familiar adenomatous polyposis (FAP), characterized by the formation of multiple colonic adenomatous polyps . Inactivation of both APC alleles (APC -/- ) is considered necessary for tumor formation. The APC protein forms a destruction complex with Axin, glycogen synthase kinase 3β (GSK3β) and casein kinase 1 (CK1) which phosphorylates β-catenin at multiple sites , and targets β-catenin for ubiquitination and to degradation by the proteasome system . A defective APC protein leads to cytoplasmic accumulation and translocation of β-catenin to the nucleus . β-catenin, originally discovered as a cadherin-binding protein, has been shown to interact with and function as a coactivator of T-cell factor/lymphocyte enhancer factor (TCF/LEF) transcription factors. Human transcription factor 4 (hTCF-4), a TCF family member that is expressed in human colonic epithelium and colon carcinoma cells, transactivates transcription only when associated with β-catenin . The result is expression and production of mitogenic and survival genes including c-Myc , cyclin D1  and cyclooxygenase-2 (COX-2) .
COX-2 levels are elevated in as many as 85% of human CRCs and approximately 50% of colorectal adenomas . Studies have shown that COX inhibition by non-steroidal anti-inflammatory drugs (NSAIDS) or aspirin reduces the risk of CRC and may be beneficial in large population groups at risk . Selective COX-2 inhibitors are also associated with a decline in the incidence of CRC and reduced mortality rate, although COX-2 inhibitors have been associated with serious cardiovascular events in this context . Prostaglandin E2 (PGE2) has been shown to be an important mediator of COX-2 associated effects, and PGE2 levels are elevated in CRC biopsies compared with normal mucosa and even in patient blood samples . Beside an anti-angiogenic effect , COX inhibition promotes apoptosis and alters tumor growth . PGE2 and COX-2 over-expression also correlates with CRC risk and metastasis of CRC , making this pathway relevant also in follow-up after treatment of the primary cancer. Furthermore, our observations show that the PGE2 produced also inhibits anti-tumor immunity through the EP2 prostanoid receptor - cAMP - protein kinase A (PKA) - Csk pathway in effector T cells that inhibit T cell activation .
Both the Wnt-Frz and the PGE2-EP3 pathway acting through phosphoinositide 3-kinase (PI3K) and protein kinase B (PKB) negatively regulates the APC destruction complex that controls β-catenin proteosomal degradation. COX inhibitors are thought to reverse the inhibitory effect of PGE2-EP3 receptor signaling on the APC destruction complex promoting β-catenin degradation and reversing the mitogenic effects. However, homozygous deletion of the gene for the PGE2 receptor EP2 also reduced the number and size of colorectal polyps in a polyposis mouse model . Furthermore, recent reports have shown that PKA can phosphorylate β-catenin at Ser552  and Ser675 [16, 17] and that the effect of β-catenin phosphorylation at the latter site is mediated by non-canonical mechanism(s) that does not involve regulation of the formation of the destruction complex. While Taurin et al. show that Ser675 phosphorylation promotes β-catenin interaction with the transcriptional coactivator CREB-binding protein in the nucleus and does not affect β-catenin stability and intracellular location , Hino et al. report that PKA phosphorylation of the same site stabilizes β-catenin and affects its intracellular localization . These differences highlight the complexity of regulation of Wnt-β-catenin signaling and may relate to the experimental conditions and system examined. Finally, PGE2 has been shown to control β-catenin homeostasis in zebrafish stem cells by signaling through both the EP3 receptor to the destruction complex and through the EP2 and EP4 receptors via cAMP to PKA affecting β-catenin stability . Given the importance of β-catenin as a trans-activator in CRC and the interest in COX chemoprevention, the question of whether the PGE2-EP2/4-cAMP-PKA pathway is also active in controlling β-catenin levels in CRC is highly relevant .
The ApcMin/+ mouse is a well-established model of FAP with a germline mutation in one APC allele, thus increasing the probability of a double allele mutation and tumor formation. ApcMin/+ mice develop multiple adenomas in the intestinal tract, mainly in the small intestine, at an early age which can be blocked effectively by COX inhibition through NSAIDS. Here, we asked whether perturbation of the EP2/4 but not the EP3 pathway by inhibition at the level of PKA, could affect β-catenin levels and tumor formation. We show that treatment of ApcMin/+ mice with a PKA antagonist, Rp-8-Br-cAMPS, reduces tumor load, β-catenin levels and nuclear translocation as well as expression of β-catenin target genes in ApcMin/+ mice in vivo.
Differential effects of COX and PKA inhibition on tumor formation in ApcMin/+ mice
Inhibition of PKA does not affect lymphocytic tumor infiltration or HIF-1α expression in ApcMin/+ mice tumors
PGE2 also affects angiogenesis and up-regulates vascular endothelial growth factor receptor-1 (VEGFR-1) in a human colon cancer cell line  whereas indomethacin inhibits the expression of VEGF and thereby angiogenesis . To assess treatments effects on angiogenesis, we examined levels of the hypoxia-inducible transcription factor (HIF)-1α which regulates the expression of target genes important in angiogenesis by accumulation and translocation to the nucleus under hypoxic conditions. While apical regions of all tumors showed higher cytoplasmic intensity and nuclear staining of HIF-1α, no differences between treatment groups were observed (Figure 2C and Additional file 2, Figure S1).
PKA antagonist treatment of ApcMin/+ mice decreases the levels β-catenin signaling to the nucleus and of COX-2 and c-Myc expression in ApcMin/+ mice tumors
Cytoplasmic β-catenin may be targeted to proteosomal degradation through the destruction complex consisting of GSK3β, Axin, CK1 and APC (Figure 4C). However, in the presence of active Wnt signaling, β-catenin accumulates in the cytosol and translocates to the nucleus to act in a mitogenic fashion by transactivation of TCF/LEF leading to expression of target genes in a cell proliferation and survival program . As is well established, the Wnt-Frz pathway inhibits the destruction complex at the level of GSK3β, leading to less proteosomal degradation and more nuclear translocation and activation of β-catenin . Similarly, the up-regulation of COX-2 in colorectal cancer leads to production of PGE2 which binds to the EP3 receptor leading to PI3K and PKB activation, phosphorylation and dissociation of GSK3β and thereby inhibition of the destruction complex  (Figure 4C). In zebrafish stem cells, PGE2 acting through an EP2/4-cAMP-PKA pathway was recently shown to induce direct phosphorylation of β-catenin, thereby stimulating its translocation to the nucleus and mitogenic effect . Here, we tested whether this second pathway was providing a mitogenic drive in intestinal cancer. Using ApcMin/+ mice with a disturbed β-catenin degradation, we specifically inhibited the PGE2-cAMP pathway at the level of PKA by treating mice with Rp-8-Br-cAMPS for 6 weeks (see, Figure 4C for point of action). We show that this not only reduces tumor load but also specifically inhibits β-catenin nuclear translocation and the activation of β-catenin target genes such as c-Myc and COX-2 which may indicate that the direct regulatory effect of PKA on β-catenin nuclear translocation is also operative in intestinal cancer cells. Furthermore, the fact that COX inhibitors may block the effect of PGE2 both in the β-catenin degradation and β-catenin nuclear translocation pathways while Rp-8-Br-cAMPS only affects the latter may explain why inhibitory effect of the PKA antagonist on tumor promotion is comparably weaker than that of indomethacin. Finally, our observation that COX inhibitor abolishes tumor numbers whereas PKA antagonist reduces tumor load but not tumor numbers may indicate that the anti-tumorigenic and anti-proliferative effects are distinct and relate to different points of action in PGE2 signal pathways. It is interesting to speculate that stem cells in crypt foci that give origin to adenomas may be more sensitive to regulation via the Wnt-Frz and PGE2-EP3 pathways than via the EP2/4-cAMP-PGE2 pathway whereas this balance may shift during tumor development.
The work was funded by the Norwegian Cancer Society and the Research Council of Norway. KWB is a fellow of the Norwegian Cancer Society. We are grateful to Dr. Mioara Andrei, Lauras AS for synthesizing gram quantities of Rp-8-Br-cAMPS provided as a gift, to Dr. Henrik Rasmussen, Victor Ong and Mona I. Skullerud, Animal Unit, Norwegian Institute of Public Health for help with animal experimentation and care, to Tove Noren, Department of Pathology, Oslo University Hospital, Ullevål for help with sectioning and immunohistochemistry, and to Jorun Solheim and Gladys Tjørhom, Biotechnology Centre of Oslo for help with Western blot analysis.
- Half E, Bercovich D, Rozen P: Familial adenomatous polyposis. Orphanet J Rare Dis. 2009, 4: 22- 10.1186/1750-1172-4-22PubMed CentralView ArticlePubMedGoogle Scholar
- Rubinfeld B, Albert I, Porfiri E, Fiol C, Munemitsu S, Polakis P: Binding of GSK3beta to the APC-beta-catenin complex and regulation of complex assembly. Science. 1996, 272: 1023-1026. 10.1126/science.272.5264.1023View ArticlePubMedGoogle Scholar
- Aberle H, Bauer A, Stappert J, Kispert A, Kemler R: beta-catenin is a target for the ubiquitin-proteasome pathway. EMBO J. 1997, 16: 3797-3804. 10.1093/emboj/16.13.3797PubMed CentralView ArticlePubMedGoogle Scholar
- Städeli R, Hoffmans R, Basler K: Transcription under the Control of Nuclear Arm/[beta]-Catenin. Current Biology. 2006, 16: R378-R385. 10.1016/j.cub.2006.04.019View ArticlePubMedGoogle Scholar
- Korinek V, Barker N, Morin PJ, van Wichen D, de Weger R, Kinzler KW, Vogelstein B, Clevers H: Constitutive Transcriptional Activation by a b-Catenin-Tcf Complex in APC-/- Colon Carcinoma. Science. 1997, 275: 1784-1787. 10.1126/science.275.5307.1784View ArticlePubMedGoogle Scholar
- He TC, Sparks AB, Rago C, Hermeking H, Zawel L, da Costa LT, Morin PJ, Vogelstein B, Kinzler KW: Identification of c-MYC as a Target of the APC Pathway. Science. 1998, 281: 1509-1512.View ArticlePubMedGoogle Scholar
- Tetsu O, McCormick F: [beta]-Catenin regulates expression of cyclin D1 in colon carcinoma cells. Nature. 1999, 398: 422-426. 10.1038/18884View ArticlePubMedGoogle Scholar
- Eberhart CE, Coffey RJ, Radhika A, Giardiello FM, Ferrenbach S, DuBois RN: Up-regulation of cyclooxygenase 2 gene expression in human colorectal adenomas and adenocarcinomas. Gastroenterology. 1994, 107: 1183-1188.PubMedGoogle Scholar
- Kraus S, Arber N: Cancer: Do aspirin and other NSAIDs protect against colorectal cancer?. Nat Rev Gastroenterol Hepatol. 2011, 8: 125-126. 10.1038/nrgastro.2010.217View ArticlePubMedGoogle Scholar
- Psaty BM, Potter JD: Risks and Benefits of Celecoxib to Prevent Recurrent Adenomas. New England Journal of Medicine. 2006, 355: 950-952. 10.1056/NEJMe068158View ArticlePubMedGoogle Scholar
- Yaqub S, Henjum K, Mahic M, Jahnsen FL, Aandahl EM, Bjornbeth BA, Tasken K: Regulatory T cells in colorectal cancer patients suppress anti-tumor immune activity in a COX-2 dependent manner. Cancer Immunol Immunother. 2008, 57: 813-821. 10.1007/s00262-007-0417-xView ArticlePubMedGoogle Scholar
- Fujino H, Toyomura K, Chen Xb, Regan JW, Murayama T: Prostaglandin E2 regulates cellular migration via induction of vascular endothelial growth factor receptor-1 in HCA-7 human colon cancer cells. Biochemical Pharmacology. 2011, 81: 379-387. 10.1016/j.bcp.2010.11.001View ArticlePubMedGoogle Scholar
- Sheng H, Shao J, Washington MK, DuBois RN: Prostaglandin E2 increases growth and motility of colorectal carcinoma cells. J Biol Chem. 2001, 276: 18075-18081. 10.1074/jbc.M009689200View ArticlePubMedGoogle Scholar
- Tsujii M, Kawano S, DuBois RN: Cyclooxygenase-2 expression in human colon cancer cells increases metastatic potential. Proc Natl Acad Sci USA. 1997, 94: 3336-3340. 10.1073/pnas.94.7.3336PubMed CentralView ArticlePubMedGoogle Scholar
- Sonoshita M, Takaku K, Sasaki N, Sugimoto Y, Ushikubi F, Narumiya S, Oshima M, Taketo MM: Acceleration of intestinal polyposis through prostaglandin receptor EP2 in Apc[Delta]716 knockout mice. Nat Med. 2001, 7: 1048-1051. 10.1038/nm0901-1048View ArticlePubMedGoogle Scholar
- Taurin S, Sandbo N, Qin Y, Browning D, Dulin NO: Phosphorylation of beta-catenin by cyclic AMP-dependent protein kinase. J Biol Chem. 2006, 281: 9971-9976. 10.1074/jbc.M508778200View ArticlePubMedGoogle Scholar
- Hino S, Tanji C, Nakayama KI, Kikuchi A: Phosphorylation of beta-catenin by cyclic AMP-dependent protein kinase stabilizes beta-catenin through inhibition of its ubiquitination. Mol Cell Biol. 2005, 25: 9063-9072. 10.1128/MCB.25.20.9063-9072.2005PubMed CentralView ArticlePubMedGoogle Scholar
- Goessling W, North TE, Loewer S, Lord AM, Lee S, Stoick-Cooper CL, Weidinger G, Puder M, Daley GQ, Moon RT: Genetic interaction of PGE2 and Wnt signaling regulates developmental specification of stem cells and regeneration. Cell. 2009, 136: 1136-1147. 10.1016/j.cell.2009.01.015PubMed CentralView ArticlePubMedGoogle Scholar
- Buchanan FG, DuBois RN: Connecting COX-2 and Wnt in cancer. Cancer Cell. 2006, 9: 6-8. 10.1016/j.ccr.2005.12.029View ArticlePubMedGoogle Scholar
- Chiu CH, McEntee MF, Whelan J: Discordant effect of aspirin and indomethacin on intestinal tumor burden inApcMin/+ mice. Prostaglandins, Leukotrienes and Essential Fatty Acids. 2000, 62: 269-275. 10.1054/plef.2000.0154.View ArticleGoogle Scholar
- Galon J, Costes A, Sanchez-Cabo F, Kirilovsky A, Mlecnik B, Lagorce-Pages C, Tosolini M, Camus M, Berger A, Wind P: Type, density, and location of immune cells within human colorectal tumors predict clinical outcome. Science. 2006, 313: 1960-1964. 10.1126/science.1129139View ArticlePubMedGoogle Scholar
- Wang RF: Immune suppression by tumor-specific CD4+ regulatory T-cells in cancer. Semin Cancer Biol. 2006, 16: 73-79. 10.1016/j.semcancer.2005.07.009View ArticlePubMedGoogle Scholar
- Kettunen HL, Kettunen AS, Rautonen NE: Intestinal immune responses in wild-type and Apcmin/+ mouse, a model for colon cancer. Cancer Res. 2003, 63: 5136-5142.PubMedGoogle Scholar
- Wang HM, Zhang GY: Indomethacin suppresses growth of colon cancer via inhibition of angiogenesis in vivo. World J Gastroenterol. 2005, 11: 340-343.PubMed CentralView ArticlePubMedGoogle Scholar
- Agarwal B, Swaroop P, Protiva P, Raj SV, Shirin H, Holt PR: Cox-2 is needed but not sufficient for apoptosis induced by Cox-2 selective inhibitors in colon cancer cells. Apoptosis. 2003, 8: 649-654.View ArticlePubMedGoogle Scholar
- Castellone MD, Teramoto H, Williams BO, Druey KM, Gutkind JS: Prostaglandin E2 promotes colon cancer cell growth through a Gs-axin-beta-catenin signaling axis. Science. 2005, 310: 1504-1510. 10.1126/science.1116221View ArticlePubMedGoogle Scholar
- Andrei M, Bjornstad V, Langli G, Romming C, Klaveness J, Tasken K, Undheim K: Stereoselective preparation of (RP)-8-hetaryladenosine-3[prime or minute], 5[prime or minute]-cyclic phosphorothioic acids. Org Biomol Chem. 2007, 5: 2070-2080. 10.1039/b702403gView ArticlePubMedGoogle Scholar
- Nayjib B, Zeddou M, Drion P, Boniver J, Tasken K, Rahmouni S, Moutschen M: In vivo administration of a PKA type I inhibitor (Rp-8-Br-cAMPS) restores T-cell responses in retrovirus-infected mice. Open Immunol J. 2008, 20-24.Google Scholar
- Koch T, Petro A, Darrabie M, Opara E: Effects of Esomeprazole Magnesium on Nonsteroidal Anti-Inflammatory Drug Gastropathy. Digestive Diseases and Sciences. 2005, 50: 86-93. 10.1007/s10620-005-1283-zView ArticlePubMedGoogle Scholar
- Paulsen JE, Steffensen IL, Andreassen A, Vikse R, Alexander J: Neonatal exposure to the food mutagen 2-amino-1-methyl-6-phenylimidazo[4, 5-b]pyridine via breast milk or directly induces intestinal tumors in multiple intestinal neoplasia mice. Carcinogenesis. 1999, 20: 1277-1282. 10.1093/carcin/20.7.1277View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.