FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2024579029> ?p ?o ?g. }

• W2024579029 endingPage "14293" @default.
• W2024579029 startingPage "14287" @default.
• W2024579029 abstract "Cyclooxygenase (COX)-generated prostaglandin E2 (PGE2) plays critical roles in colorectal carcinogenesis. Recently, we have shown that PGE2 and transforming growth factor-α synergistically induces the expression of amphiregulin (AR) in colon cancer cells (Shao, J., Evers, B. M., and Sheng, H. (2003) Cancer Res. 63, 5218-5223). In this study, we demonstrated synergistic actions of PGE2 and the receptor tyrosine kinase signaling system in AR expression and in tumorigenic potential of colon cancer cells. Activation of the Ras/Raf/MAPK pathway induced AR transcription in colon cancer LS-174 cells that was enhanced by PGE2 in a synergistic fashion. The cAMP-responsive element within the AR promoter was required for the synergistic activation of AR transcription. An Sp1 element was responsible for the basal transcription of AR and significantly enhanced the synergy between PGE2 and the epidermal growth factor receptor (EGFR) signaling system. Furthermore, activation of both PGE2 and EGFR signaling pathways synergistically promoted the growth and migration of colon cancer cells. Our results suggest that COX-2/PGE2 may exert pro-oncogenic effects through synergistic induction of receptor tyrosine kinase-dependent signaling pathway, thus, provide a novel mechanism for the combinatorial treatment of colonic neoplasms targeting both COX-2/PGE2 and the EGFR system that has demonstrated remarkable advantages. Cyclooxygenase (COX)-generated prostaglandin E2 (PGE2) plays critical roles in colorectal carcinogenesis. Recently, we have shown that PGE2 and transforming growth factor-α synergistically induces the expression of amphiregulin (AR) in colon cancer cells (Shao, J., Evers, B. M., and Sheng, H. (2003) Cancer Res. 63, 5218-5223). In this study, we demonstrated synergistic actions of PGE2 and the receptor tyrosine kinase signaling system in AR expression and in tumorigenic potential of colon cancer cells. Activation of the Ras/Raf/MAPK pathway induced AR transcription in colon cancer LS-174 cells that was enhanced by PGE2 in a synergistic fashion. The cAMP-responsive element within the AR promoter was required for the synergistic activation of AR transcription. An Sp1 element was responsible for the basal transcription of AR and significantly enhanced the synergy between PGE2 and the epidermal growth factor receptor (EGFR) signaling system. Furthermore, activation of both PGE2 and EGFR signaling pathways synergistically promoted the growth and migration of colon cancer cells. Our results suggest that COX-2/PGE2 may exert pro-oncogenic effects through synergistic induction of receptor tyrosine kinase-dependent signaling pathway, thus, provide a novel mechanism for the combinatorial treatment of colonic neoplasms targeting both COX-2/PGE2 and the EGFR system that has demonstrated remarkable advantages. A large body of studies indicates that cyclooxygenase-2 (COX-2) 1The abbreviations used are: COX, cyclooxygenase; PGE2, prostaglandin E2; PGs, prostaglandins; TGF-α, transforming growth factor α; EGFR, epidermal growth factor receptor; RTK, receptor tyrosine kinase; AR, amphiregulin; PKA, protein kinase A; ANOVA, analysis of variance; ERK, extracellular signal-regulated kinase; CRE, cAMP-responsive element; MEK, MAPK/ERK kinase; MAPK, mitogen-activated protein kinase; CREB, cAMP-response element-binding protein; EP, E-type prostaglandin receptor; Rsk, ERK/p90rsk. and its derived prostaglandin E2 (PGE2) exert pro-oncogenic effects on colorectal neoplasms (1Gupta R.A. Dubois R.N. Nat. Rev. Cancer. 2001; 1: 11-21Google Scholar). Disruption of key steps of the metabolism of arachidonic acid to prostaglandins (PGs) results in tumor reduction in ApcΔ716 mice. For examples, genetic deletion of cytosolic phospholipase A2, which releases arachidonic acid from cell membrane, or knock-out of COX-2 gene, which encodes the key enzyme for conversion of arachidonic acid to PGs, results in an ∼60% reduction of Apc mutation-induced intestinal adenomas in mice (2Oshima M. Dinchuk J.E. Kargman S. Oshima H. Hancock B. Kwong E. Trzaskos J.M. Evans J.F. Taketo M.M. Cell. 1996; 87: 803-809Google Scholar, 3Takaku K. Sonoshita M. Sasaki N. Uozumi N. Doi Y. Shimizu T. Taketo M.M. J. Biol. Chem. 2000; 275: 34013-34016Google Scholar). In addition, disruption of E-type prostaglandin receptor, EP2, which mediates PGE2 signaling, reduces the number of adenomas by ∼60% in ApcΔ716 mice as well (4Sonoshita M. Takaku K. Sasaki N. Sugimoto Y. Ushikubi F. Narumiya S. Oshima M. Taketo M.M. Nat. Med. 2001; 7: 1048-1051Google Scholar), suggesting the critical role of the PGE2 signaling pathway in intestinal neoplasia. PGE2 promotes proliferation of human colorectal carcinoma cells (5Pai R. Soreghan B. Szabo I.L. Pavelka M. Baatar D. Tarnawski A.S. Nat. Med. 2002; 8: 289-293Google Scholar). We have shown that PGE2 stimulates the growth of human colorectal cancer cells when grown in extracellular matrix (6Sheng H. Shao J. Kirkland S.C. Isakson P. Coffey R.J. Morrow J.D. Beauchamp R.D. DuBois R.N. J. Clin. Investig. 1997; 99: 2254-2259Google Scholar, 7Sheng H. Shao J. Washington M.K. DuBois R.N. J. Biol. Chem. 2001; 276: 18075-18081Google Scholar, 8Shao J. Lee S.B. Guo H. Evers B.M. Sheng H. Cancer Res. 2003; 63: 5218-5223Google Scholar). In addition, PGE2 promotes colon cancer cell migration and increases their metastatic potential (7Sheng H. Shao J. Washington M.K. DuBois R.N. J. Biol. Chem. 2001; 276: 18075-18081Google Scholar, 9Tsujii M. Sunao K. DuBois R.N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3336-3340Google Scholar, 10Buchanan F.G. Wang D. Bargiacchi F. DuBois R.N. J. Biol. Chem. 2003; 278: 35451-35457Google Scholar, 11Pai R. Nakamura T. Moon W.S. Tarnawski A.S. FASEB J. 2003; 17: 1640-1647Google Scholar). PGE2 acts via four PGE receptor (EP) subtypes, which are designated as EP1, EP2, EP3, and EP4 (12Breyer M.D. Breyer R.M. Curr. Opin. Nephrol. Hypertens. 2000; 9: 23-29Google Scholar). EP2 and EP4 receptors signal through increased cAMP and are thought to mediate PGE2 pro-oncogenic actions (4Sonoshita M. Takaku K. Sasaki N. Sugimoto Y. Ushikubi F. Narumiya S. Oshima M. Taketo M.M. Nat. Med. 2001; 7: 1048-1051Google Scholar, 7Sheng H. Shao J. Washington M.K. DuBois R.N. J. Biol. Chem. 2001; 276: 18075-18081Google Scholar, 8Shao J. Lee S.B. Guo H. Evers B.M. Sheng H. Cancer Res. 2003; 63: 5218-5223Google Scholar). Thus far, the molecular mechanisms that mediate tumor-promoting effects of the PGE2 signaling system have not been extensively investigated. The epidermal growth factor family and their cognate receptors (EGFR), referred to as the ErbB family, play critical roles in colorectal carcinogenesis (13Karnes Jr., W.E. Weller S.G. Adjei P.N. Kottke T.J. Glenn K.S. Gores G.J. Kaufmann S.H. Gastroenterology. 1998; 114: 930-939Google Scholar, 14Roh H. Pippin J. Drebin J.A. Cancer Res. 2000; 60: 560-565Google Scholar, 15Oldham S.M. Cox A.D. Reynolds E.R. Sizemore N.S. Coffey R.J.J. Der C.J. Oncogene. 1998; 16: 2565-2573Google Scholar, 16Sheng H. Shao H. DuBois R.N. J. Biol. Chem. 2001; 276: 14498-14504Google Scholar). Binding of the ligand to the EGFR leads to activation of receptor tyrosine kinases (RTKs) that phosphorylate tyrosine residues of cellular signaling proteins and activate signaling pathways that are essential for intestinal epithelial proliferation and transformation (17Podolsky D.K. Johnson L.R. Physiology of the Gastrointestinal Tract. 3rd Ed. Raven Press, Ltd., New York1994: 129-167Google Scholar, 18Jones M.K. Tomikawa M. Mohajer B. Tarnawski A.S. Front. Biosci. 1999; 4: D303-D309Google Scholar, 19Polk D.B. Barnard J.A. Sanderson I.R. Walker A. Development of the Gastrointestinal Tract. B. C. Decker Inc., Hamilton, Ontario, Canada1999: 37-55Google Scholar). PGE2 transactivates the EGFR, triggers extracellular signaling-regulated kinase (ERK) activation, and stimulates the proliferation of colorectal carcinoma cells (5Pai R. Soreghan B. Szabo I.L. Pavelka M. Baatar D. Tarnawski A.S. Nat. Med. 2002; 8: 289-293Google Scholar, 20Sheng H. Shao J. Morrow J.D. Beauchamp R.D. DuBois R.N. Cancer Res. 1998; 58: 362-366Google Scholar). The phosphatidylinositol 3-kinase/Akt pathway transmits signals from tyrosine kinase-coupled receptors and plays critical roles in mitogenic signaling (21Katso R. Okkenhaug K. Ahmadi K. White S. Timms J. Waterfield M.D. Annu. Rev. Cell Dev. Biol. 2001; 17: 615-675Google Scholar). Phosphatidylinositol 3-kinase activity that is critical for the proliferation and transformation of intestinal epithelial cells (16Sheng H. Shao H. DuBois R.N. J. Biol. Chem. 2001; 276: 14498-14504Google Scholar, 22Sheng H. Shao J. Townsend Jr., C.M. Evers B.M. Gut. 2003; 52: 1472-1478Google Scholar) is required for the growth stimulation of PGE2 in human colon cancer cells (7Sheng H. Shao J. Washington M.K. DuBois R.N. J. Biol. Chem. 2001; 276: 18075-18081Google Scholar). PGE2-induced DNA synthesis can be blocked by specific EGFR antagonists or antibodies to transforming growth factor α (TGF-α) and amphiregulin (5Pai R. Soreghan B. Szabo I.L. Pavelka M. Baatar D. Tarnawski A.S. Nat. Med. 2002; 8: 289-293Google Scholar, 8Shao J. Lee S.B. Guo H. Evers B.M. Sheng H. Cancer Res. 2003; 63: 5218-5223Google Scholar). Recently, we have reported that PGE2 induces the expression of amphiregulin (AR), a member of the epidermal growth factor family, which exerts mitogenic effect to colon cancer cells through an autocrine mechanism (8Shao J. Lee S.B. Guo H. Evers B.M. Sheng H. Cancer Res. 2003; 63: 5218-5223Google Scholar). We have demonstrated that PGE2 and TGF-α induced the expression of AR mRNA in a synergistic manner. These studies indicate the complexity of interaction between PGE2 and the EGFR signaling system and suggest that PGE2 may activate RTK-dependent signaling pathways through a variety of mechanisms. COX inhibitors are being developed as agents for the intervention of colorectal cancers (1Gupta R.A. Dubois R.N. Nat. Rev. Cancer. 2001; 1: 11-21Google Scholar). Elucidating the interaction between the PGE2 signaling pathway and other oncogenic signaling systems further will help to design novel strategies for prevention and treatment of colon cancers. In this study, we elucidated the mechanism by which PGE2 and the EGFR/Ras/MAPK system synergistically induced the expression of pro-oncogenic gene, AR. The functional synergy of PGE2 and the EGFR was elucidated. PGE2 and TGF-α stimulated the growth and migration of colon cancer cells in a synergistic manner. Thus, our results provide a novel mechanism for combined treatment of colorectal neoplasia by targeting both the COX-2/PGE2 pathway and the RTK signaling system. Cell Culture and Chemicals—LS-174 and T-84 human colon cancer cell lines were purchased from ATCC (Manassas, VA). LS-174 cells were maintained in McCoy's 5A medium containing 10% fetal bovine serum. T-84 cells were maintained in Dulbecco's modified Eagle's medium/F12 medium containing 5% fetal bovine serum. Growth of cells in Matrigel® (Collaborative Biomedical, Bedford, MA) was carried out as described previously (7Sheng H. Shao J. Washington M.K. DuBois R.N. J. Biol. Chem. 2001; 276: 18075-18081Google Scholar). PGE2 was purchased from Cayman Chemical (Ann Arbor, MI). TGF-α was purchased from R&D system (Minneapolis, MN). H-89 and PD-153035 were purchased from Calbiochem. Dibutyryl cAMP was purchased from Sigma. Cell Migration Assays—Cell migration assay was carried out using Transwell chambers (8 μm, Corning Costar Co. Cambridge, MA). 5 × 104 cells suspended in 400 μl of serum-free McCoy's 5A medium were placed in the upper chamber. The lower chamber was filled with 1 ml of McCoy's 5A medium containing vehicle or indicated treatment. After an incubation period of 24 h at 37 °C, the cells on the upper surface of the filter were removed with a cotton swab. The filters were fixed and stained with 0.5% crystal violet solution. Cells adhering to the undersurface of the filter were counted. Transient Transfection and Luciferase Assay—The assays to determine the activity of the AR promoter were described previously (8Shao J. Lee S.B. Guo H. Evers B.M. Sheng H. Cancer Res. 2003; 63: 5218-5223Google Scholar). Reporter constructs pGL2-A, pGL2-B, pGL2-BΔCRE, pGL2-C, and pGL2-CΔCRE containing the 5′-flanking region of the human AR gene were described previously (8Shao J. Lee S.B. Guo H. Evers B.M. Sheng H. Cancer Res. 2003; 63: 5218-5223Google Scholar, 24Lee S.B. Huang K. Palmer R. Truong V.B. Herzlinger D. Kolquist K.A. Wong J. Paulding C. Yoon S.K. Gerald W. Oliner J.D. Haber D.A. Cell. 1999; 98: 663-673Google Scholar). pGL2-A1 (-417 to -192) and pGL2-A2 (-372 to -192) were constructed using PCR. pGL2-A2ΔCRE was generated by digestion of pGL2-A2 with AatII and treatment with T4 DNA polymerase. For transient transfections, cells were co-transfected with indicated plasmids including 3 ng of the pRL-SV40 plasmid containing the Renilla luciferase gene (Promega, Madison WI) using the FuGENE 6 procedure (Roche Applied Science). Firefly and Renilla luciferase activities were measured using a dual-luciferase reporter assay system (Promega) and a luminometer. Firefly luciferase values were standardized to Renilla values and expressed as relative light units. The mammalian expression vector pSRα/Raf BXB contains the BXB mutant of c-Raf-1 kinase lacking amino acids 26-203 (kindly provided by Dr. Michael White). The active MEK1 expression vector, pMEK1(S218D/S222D), and wild type MEK1 expression vector, pMEK1, were purchased from Upstate Biotechnology (Lake Placid, NY). The active CREB expression vector, pCMV-CREB, and the dominant negative CREB, pCMV-KCREB, were purchased form BD Biosciences. Immunoblot Analysis—Immunoblot analysis was performed as described previously (23Shao J. Sheng H. Inoue H. Morrow J.D. DuBois R.N. J. Biol. Chem. 2000; 275: 33951-33956Google Scholar). Cells were lysed for 30 min in radioimmune precipitation assay buffer (1× phosphate-buffered saline, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 mg/ml phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 1 mm sodium orthovanadate) and then clarified cell lysates were denatured and fractionated by SDS-PAGE. After electrophoresis, the proteins were transferred to nitrocellulose membranes and then the filters were incubated with the antibodies indicated and developed by the ECL system (Amersham Biosciences). The anti-pCREB, anti-pERK, and anti-pRSK antibodies were purchased from Cell Signaling (Beverly, MA). Data Analysis—All of the statistical analyses were performed on a personal computer with the StatView, version 5.0.1 software (SAS Institute Inc. Cary, NC). Analyses among multiple groups were determined by ANOVA. Analyses between two groups were determined using the unpaired Student's t test. Differences with a p value of <0.05 were considered as statistically significant. Synergy between PGE2 and the Ras/Raf/MAPK Pathway—Cell growth and transformation require the expression of a subset of pro-oncogenic genes. Amphiregulin plays critical roles in colon cancer cell proliferation and transformation (25De Luca A. Arra C. D'Antonio A. Casamassimi A. Losito S. Ferraro P. Ciardiello F. Salomon D.S. Normanno N. Oncogene. 2000; 19: 5863-5871Google Scholar, 26Damstrup L. Kuwada S.K. Dempsey P.J. Brown C.L. Hawkey C.J. Poulsen H.S. Wiley H.S. Coffey Jr., R.J. Br. J. Cancer. 1999; 80: 1012-1019Google Scholar). We have demonstrated that PGE2 and TGF-α increase the expression of AR mRNA through synergistic induction of AR transcription (8Shao J. Lee S.B. Guo H. Evers B.M. Sheng H. Cancer Res. 2003; 63: 5218-5223Google Scholar). The Ras/Raf/MAPK pathway plays a central role in transduction of signals from EGFR. PGE2 and oncogenic K-Ras induces AR transcription in a synergistic manner (8Shao J. Lee S.B. Guo H. Evers B.M. Sheng H. Cancer Res. 2003; 63: 5218-5223Google Scholar). To completely assess the interaction between PGE2 and the Ras/Raf/MAPK pathway, we ectopically introduced mutated Raf and MAPK/ERK kinase (MEK) into LS-174 cells. Transfection with an active Raf expression vector, Raf-BXB (16Sheng H. Shao H. DuBois R.N. J. Biol. Chem. 2001; 276: 14498-14504Google Scholar), increased AR transcription ∼2.5-fold. The addition of PGE2 resulted in a 19-fold increase in the activity of AR promoter when compared with the cells transfected with empty vector (Fig. 1A). In contrast, expression of a kinase-dead Raf construct did not change AR transcription. Moreover, expression of an active form of MEK1 increased AR promoter activity ∼13-fold. Again, a synergistic induction was observed between PGE2 and MEK1 activity (Fig. 1B). These results suggest a synergy between PGE2 signaling and the Ras/Raf/MAPK pathway in context of AR transcription. TGF-α signals through the EGFR and RTK-dependent signaling pathways. PGE2 acts through induction of cAMP and activation of protein kinase A (PKA). Inhibition of either EGFR tyrosine kinases or the cAMP/PKA pathway attenuated the synergy of TGF-α and PGE2. Either a selective PKA inhibitor, H-89, or a specific EGFR tyrosine kinase inhibitor, PD-153035, partially attenuated the synergistic action of PGE2 and TGF-α. Combined treatment with H-89 and PD-153035 completely blocked the induction of AR transcription by PGE2 and TGF-α (Fig. 1C). Roles of the Sp1 in AR Transcription—Human AR promoter contains a number of functional domains, as schematically demonstrated in Fig. 2A (lower panel). To determine the relative contribution of each transcriptional element in AR transcription, progressive deletion constructs were generated. The luciferase activity observed with reporter vectors pGL2-A, pGL2-A1, and pGL2-A2 was approximately 20 times higher than that with pGL2 vector (Fig. 2A, upper panel). The major promoter activity was mapped to 44 nucleotides between -372 and -328 where an Sp1 consensus sequence (-353 to -348) was identified. Removal of the Sp1 site dramatically reduced AR promoter activity. Luciferase activity was decreased by ∼92% in pGL2-B-transfected cells. The reporter vector pGL2-A2, which contains the AR promoter sequence from -372 to -192 including the Sp1, a Wilms' tumor suppressor WT1-responsive element consensus, a CRE, and a TATA box, was constructed. PGE2 and TGF-α synergistically induced the luciferase activity in pGL2-A2-transfected LS-174 cells, suggesting these cis-elements are sufficient for the PGE2 and TGF-α synergy on AR transcription (Fig. 2B). Critical Roles of CRE in the PGE2 and TGF-α Synergy—The CRE element within AR promoter is critical for PGE2 induction of AR transcription (8Shao J. Lee S.B. Guo H. Evers B.M. Sheng H. Cancer Res. 2003; 63: 5218-5223Google Scholar). To evaluate the role of the Sp1 and the CRE in PGE2/TGF-α synergistic induction of AR transcription, the CRE element was mutated in pGL2-A2 construct and referred to as pGL2-A2ΔCRE. Although the basal activity of pGL2-A2 and pGL2-A2ΔCRE was similar, PGE2 did not increase the luciferase activity in pGL2-A2ΔCRE-transfected LS-174 cells. The synergistic effect of PGE2 and TGF-α was also attenuated by disruption of the CRE (Fig. 3A). Furthermore, induction of AR transcription by oncogenic K-Ras required the CRE domain. The synergistic induction of AR transcription by oncogenic K-Ras and PGE2 was attenuated when LS-174 cells were transiently transfected with pGL2-A2ΔCRE reporter construct (Fig. 3B). To confirm the critical role of the CRE in AR transcription, we co-transfected LS-174 cells with a wild type CREB expression vector and pGL2-A reporter vector. Expression of wild type CREB increased the level of luciferase activity 9-fold (Fig. 3C). In contrast, expression of a dominant negative form of CREB reduced the basal AR transcription and significantly blocked PGE2 activation of the AR promoter as well. In agreement with these observations, the addition of cAMP significantly increased AR promoter activity. A synergistic induction of AR transcription was noted when LS-174 cells were treated with both cAMP and TGF-α (Fig. 3D). The MEK-induced AR transcription was synergistically enhanced by cAMP treatment as well. To further determine the critical role of the CRE in PGE2/TGF-α regulation of AR transcription, LS-174 cells were transfected with pGL2-B reporter vector, which contains a Wilms' tumor suppressor WT1-responsive element, the CRE, and a TATA box. Removal of the Sp1 element significantly reduced AR promoter activity. The basal activity of pGL2-B was only ∼10% of the activity of the pGL2-A2; however, the PGE2/TGF-α-mediated regulation of AR promoter activity was fully preserved in pGL2-B-transfected LS-174 cells (Fig. 4A). PGE2 induced pGL2-B activity 9-fold, and PGE2 and TGF-α synergistically increased pGL2-B activity 19-fold. Mutation of the CRE completely attenuated the activity of the AR promoter. Similar results were observed in LS-174 cells that were transfected with pGL2-C promoter construct, which contains only 83 nucleotides (-275 to -192) including the CRE and a TATA box (Fig. 4B). We have demonstrated that PGE2 treatment rapidly induces the phosphorylation of CREB at Ser-133. Several studies indicate that TGF-α may induce phosphorylation and activation of CREB through the ERK/p90rsk (Rsk) pathway (28De Cesare D. Jacquot S. Hanauer A. Sassone-Corsi P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12202-12207Google Scholar, 29Dalby K.N. Morrice N. Caudwell F.B. Avruch J. Cohen P. J. Biol. Chem. 1998; 273: 1496-1505Google Scholar). Western analysis revealed that TGF-α treatment increased levels of phosphorylated ERK, phosphorylated Rsk, and phosphorylated CREB (Ser-133) in LS-174 cells (Fig. 4C). PGE2 and TGF-α Synergistically Stimulated Colon Cancer Cell Growth—Because the EGFR signaling system and the COX-2/PGE2 signaling system can synergistically induce pro-oncogenic gene expression, it was of interest to determine whether these two signaling systems collaboratively enhance tumorigenic potential. We first elucidated the growth regulatory effects of PGE2 and TGF-α in colon cancer cells. LS-174 cells are able to form “tumor-like” aggregates when they are cultured in Matrigel (7Sheng H. Shao J. Washington M.K. DuBois R.N. J. Biol. Chem. 2001; 276: 18075-18081Google Scholar). The colonies consisted of several layers of cells and a mucin-filled center. Morphologically, colonies formed by LS-174 cells in Matrigel were highly similar to LS-174 xenografts in athymic nude mice (Fig. 5A). The addition of TGF-α or PGE2 increased the size of colonies. In combination, TGF-α and PGE2 synergistically exerted growth-stimulatory effects on LS-174 cells (Fig. 5B). Colon cancer T-84 cells, which have been reported to react to PGE2 treatment (30Yu Y. Chadee K. J. Immunol. 1998; 161: 3746-3752Google Scholar), formed similar three-dimensional structures in Matrigel. The synergistic stimulation of TGF-α and PGE2 on cell growth was observed in T-84 cells as well (Fig. 5C). These results suggest that TGF-α and PGE2 may stimulate the growth of colon tumors in a synergistic manner. PGE2 and TGF-α Synergistically Stimulated Colon Cancer Cell Migration—Several studies have demonstrated that PGE2 promotes migration and invasion of colon cancer cells (7Sheng H. Shao J. Washington M.K. DuBois R.N. J. Biol. Chem. 2001; 276: 18075-18081Google Scholar, 10Buchanan F.G. Wang D. Bargiacchi F. DuBois R.N. J. Biol. Chem. 2003; 278: 35451-35457Google Scholar, 11Pai R. Nakamura T. Moon W.S. Tarnawski A.S. FASEB J. 2003; 17: 1640-1647Google Scholar). On the other hand, TGF-α-activated EGFR signaling is critical for colon cancer cell migration (31Wilson A.J. Gibson P.R. Exp. Cell Res. 1999; 250: 187-196Google Scholar). To elucidate the collaborative regulation of cell migration by PGE2 and TGF-α, LS-174 cells were subjected to the Modified Boyden chamber assay (Transwell). Vehicle-treated LS-174 cells formed non-invasive clumps on the membrane after a 24-h incubation, so that most micropores were not covered by cells. LS-174 cells strongly spread when both PGE2 and TGF-α were present in the bottom chamber (data not shown). As a result, PGE2 and TGF-α synergistically stimulated LS-174 cell migration (Fig. 6A) and increased the number of cells that penetrated the membrane ∼15-fold (Fig. 6B). Similar results were reproduced in colon cancer T-84 cells (Fig. 6C). Colorectal cancers typically develop over decades and require a number of genetic changes for completion (32Kinzler K.W. Vogelstein B. Cell. 1996; 87: 159-170Google Scholar). Epidemiological studies demonstrate a 40-50% reduction in the relative risk of colorectal cancer and colorectal cancer-associated mortality in individuals taking nonsteroidal anti-inflammatory drugs on a regular basis (33Thun M.J. Namboodiri M.M. Calle E.E. Flanders W.D. Heath C.W.J. Cancer Res. 1993; 53: 1322-1327Google Scholar, 34Peleg I.I. Maibach H.T. Brown S.H. Wilcox C.M. Arch. Int. Med. 1994; 154: 394-399Google Scholar, 35Giovannucci E. Egan K.M. Hunter D.J. Stampfer M.J. Colditz G.A. Willett W.C. Speizer F.E. N. Eng. J. Med. 1995; 333: 609-614Google Scholar). Genetic disruption of the COX-2/PGE2 pathway results in a ∼ 60% tumor reduction in ApcΔ716 mice (2Oshima M. Dinchuk J.E. Kargman S. Oshima H. Hancock B. Kwong E. Trzaskos J.M. Evans J.F. Taketo M.M. Cell. 1996; 87: 803-809Google Scholar, 4Sonoshita M. Takaku K. Sasaki N. Sugimoto Y. Ushikubi F. Narumiya S. Oshima M. Taketo M.M. Nat. Med. 2001; 7: 1048-1051Google Scholar). These observations indicate the critical role of COX-2/PGE2 in colon cancer development and also raised an important question. What is the molecular mechanism that mediates the chronic pro-oncogenic action of the COX-2/PGE2 signaling pathway in colorectal neoplasia? Recent studies have demonstrated that PGE2 induces rapid phosphorylation and transactivation of the EGFR through Src-mediated signaling (5Pai R. Soreghan B. Szabo I.L. Pavelka M. Baatar D. Tarnawski A.S. Nat. Med. 2002; 8: 289-293Google Scholar, 10Buchanan F.G. Wang D. Bargiacchi F. DuBois R.N. J. Biol. Chem. 2003; 278: 35451-35457Google Scholar, 11Pai R. Nakamura T. Moon W.S. Tarnawski A.S. FASEB J. 2003; 17: 1640-1647Google Scholar). Transactivation of the EGFR is particularly critical for PGE2-induced growth and invasion of colon cancer cells. Colorectal neoplasms are frequently associated with alterations of the EGFR signaling system. Epidermal growth factor receptors including their non-ligand-binding co-receptor, ErbB-2, are often up-regulated in colorectal neoplasms (36Shirai H. Ueno E. Osaki M. Tatebe S. Ito H. Kaibara N. Anticancer Res. 1995; 15: 2889-2894Google Scholar, 37Ozgul C. Karaoz E. Erdogan D. Dursun A. Acta Physiol. Hung. 1997; 85: 121-128Google Scholar). Overexpression of EGFR ligands including TGF-α, amphiregulin, and cripto in colonic tumors is well documented (25De Luca A. Arra C. D'Antonio A. Casamassimi A. Losito S. Ferraro P. Ciardiello F. Salomon D.S. Normanno N. Oncogene. 2000; 19: 5863-5871Google Scholar, 36Shirai H. Ueno E. Osaki M. Tatebe S. Ito H. Kaibara N. Anticancer Res. 1995; 15: 2889-2894Google Scholar, 38Normanno N. Selvam M.P. Bianco C. Damiano V. de Angelis E. Grassi M. Magliulo G. Tortora G. Salomon D.S. Ciardiello F. Int. J. Cancer. 1995; 62: 762-766Google Scholar). Transactivation of the EGFR by COX-generated PGE2 may further increase the activity of the EGFR and, therefore, enhance the oncogenicity of the EGFR signaling system. However, additional mechanisms appear to be necessary for the understanding of the key roles of COX-2/PGE2 in colorectal neoplasia that have been observed in epidemiological studies and in animal experiments as well (2Oshima M. Dinchuk J.E. Kargman S. Oshima H. Hancock B. Kwong E. Trzaskos J.M. Evans J.F. Taketo M.M. Cell. 1996; 87: 803-809Google Scholar, 4Sonoshita M. Takaku K. Sasaki N. Sugimoto Y. Ushikubi F. Narumiya S. Oshima M. Taketo M.M. Nat. Med. 2001; 7: 1048-1051Google Scholar, 33Thun M.J. Namboodiri M.M. Calle E.E. Flanders W.D. Heath C.W.J. Cancer Res. 1993; 53: 1322-1327Google Scholar, 34Peleg I.I. Maibach H.T. Brown S.H. Wilcox C.M. Arch. Int. Med. 1994; 154: 394-399Google Scholar, 35Giovannucci E. Egan K.M. Hunter D.J. Stampfer M.J. Colditz G.A. Willett W.C. Speizer F.E. N. Eng. J. Med. 1995; 333: 609-614Google Scholar). Our results show that the COX-2/PGE2 signaling pathway and the RTK-dependent signaling system promoted the growth and migration of colon cancer cells in a synergistic fashion. These two signaling systems synergistically induced the expression of AR, providing the evidence that COX-2/PGE2 may collaboratively enhance the expression of pro-oncogenic genes, which are regulated by growth factors and oncogenes and which are critical for neoplastic transformation of intestinal epithelium. Thus far, COX-2/PGE2 itself is not considered as an oncogenic signaling pathway; however, our results suggest that COX-2/PGE2 may dramatically increase the activity of major oncogenic signaling systems and exert pro-oncogenic actions to colonic neoplasms. Cumulative evidence suggests that COX-2 is a target gene of oncogenic Ras (39Subbaramaiah K. Telang N. Ramonetti J.T. Araki R. DeVito B. Weksler B.B. Dannenberg A.J. Cancer Res. 1996; 56: 4424-4429Google Scholar, 40Sheng H. Williams C.S. Shao J. Liang P. DuBois R.N. Beauchamp R.D. J. Biol. Chem. 1998; 273: 22120-22127Google Scholar, 41Sheng H. Shao J. DuBois R.N. Cancer Res. 2001; 61: 2670-2675Google Scholar). Inhibition of COX-2 enzyme activity suppresses the growth of xenografts of Ras-transformed rat intestinal epithelial cells in nude mice, suggesting the involvement of COX-2 in Ras-mediated transformation (42Sheng G.G. Shao J. Sheng H. Hooton E.B. Isakson P.C. Morrow J.D. Coffey R.J. DuBois R.N. Beauchamp R.D. Gastroenterology. 1997; 113: 1883-1891Google Scholar). We have reported that COX-2-generated prostacyclin enhances Ras-mediated activation of peroxisome proliferator-activated receptor δ (43Shao J. Sheng H. DuBois R.N. Cancer Res. 2002; 62: 3282-3288Google Scholar), which has been shown to protect colon cancer cells from apoptosis (44He T.C. Chan T.A. Vogelstein B. Kinzler K.W. Cell. 1999; 99: 335-345Google Scholar). In this study, we found that PGE2 synergistically enhanced Ras-induced transcription of a pro-oncogenic gene, AR, of which overexpression and involvement in colorectal tumors are well investigated (25De Luca A. Arra C. D'Antonio A. Casamassimi A. Losito S. Ferraro P. Ciardiello F. Salomon D.S. Normanno N. Oncogene. 2000; 19: 5863-5871Google Scholar, 38Normanno N. Selvam M.P. Bianco C. Damiano V. de Angelis E. Grassi M. Magliulo G. Tortora G. Salomon D.S. Ciardiello F. Int. J. Cancer. 1995; 62: 762-766Google Scholar). These findings suggest that COX-2-generated PGs may contribute to Ras-mediated transformation of intestinal epithelial cells through different mechanisms. Given the critical role of K-ras oncogene in the adenoma to carcinoma sequence of events involved in the neoplastic transformation of colonic epithelial cells, our results add new insight to the pro-oncogenic roles of the COX-2/PGs signaling system in the genetic pathway of colorectal neoplasia. The serine/threonine kinase Raf is a downstream effector of Ras (45Campbell S.L. Khosravi-Far R. Rossman K.L. Clark G.J. Der C.J. Oncogene. 1998; 17: 1395-1413Google Scholar). Activation of Raf is necessary and sufficient for the activation of the MEK/ERK cascade. Activated ERK phosphorylates and activates the Elk-1 transcription factor, thereby stimulating the transcription of genes controlled by the serum response element (46Gille H. Kortenjann M. Thomae O. Moomaw C. Slaughter C. Cobb M.H. Shaw P.E. EMBO J. 1995; 14: 951-962Google Scholar). The Raf/MEK/ERK pathway plays an important role in the control of metabolic processes, cell cycle, cell migration, cell proliferation, and cell transformation (47Schlessinger J. Cell. 2000; 103: 211-225Google Scholar) and can be activated by a large number of growth factors and hormones through Ras-dependent or Ras-independent mechanisms (48Peyssonnaux C. Eychene A. Biol. Cell. 2001; 93: 53-62Google Scholar). Our results demonstrate a direct collaboration among PGE2, Raf and PGE2, and MEK1 in regulation of AR transcription, suggesting that PGE2 is able to modulate the Raf/MEK/ERK pathway at different levels and thereby regulate a wide variety of cellular functions. The human AR promoter contains a number of functional domains (24Lee S.B. Huang K. Palmer R. Truong V.B. Herzlinger D. Kolquist K.A. Wong J. Paulding C. Yoon S.K. Gerald W. Oliner J.D. Haber D.A. Cell. 1999; 98: 663-673Google Scholar, 27Plowman G.D. Green J.M. McDonald V.L. Neubauer M.G. Disteche C.M. Todaro G.J. Shoyab M. Mol. Cell. Biol. 1990; 10: 1969-1981Google Scholar). We demonstrate that the activation of the AR promoter was controlled by a constitutive and a regulatory domain. The Sp1 element appeared to provide the constitutive activity of the AR promoter, whereas the CRE was responsible for the regulatory activation of the promoter. Deletion of the Sp1 sequence reduced AR promoter activity by 90% but preserved its reactivity to PGE2 and TGF-α stimulation. In contrast, disruption of the CRE domain maintained the basal activity of AR promoter but attenuated the PGE2/TGF-α-induced AR transcription. Our data suggest that the cAMP/PKA pathway clearly mediated PGE2 signaling and was required for PGE2 induction of AR transcription. On the other hand, the effect of TGF-α on AR transcription was modest. TGF-α treatment resulted in phosphorylation of ERK, Rsk, and CREB, suggesting that TGF-α may regulate AR transcription via CRE as well (28De Cesare D. Jacquot S. Hanauer A. Sassone-Corsi P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12202-12207Google Scholar, 29Dalby K.N. Morrice N. Caudwell F.B. Avruch J. Cohen P. J. Biol. Chem. 1998; 273: 1496-1505Google Scholar). Additional experiments are required to determine the precise mechanism by which PGE2 and TGF-α synergistically induce AR transcription. Combination of anti-cancer agents that target several genetic or biochemical alterations involved in neoplasms may result in better therapeutic response than single treatment. Torrance et al. (49Torrance C.J. Jackson P.E. Montgomery E. Kinzler K.W. Vogelstein B. Wissner A. Nunes M. Frost P. Discafani C.M. Nat. Med. 2000; 6: 1024-1028Google Scholar) report that a combination of a nonsteroidal anti-inflammatory drug, sulindac, and an EGFR kinase inhibitor, EKI-569, achieved remarkable protection from intestinal neoplasia in ApcMin/+ mice. Although untreated ApcMin/+ mice developed ∼20 polyps/mouse, EKI-569 alone reduced intestinal polyps by 50% and sulindac alone inhibited the number of polyps by 70%. Interestingly, nearly half of the mice treated with these two agents developed no polyps at all. In another study (50Mann M. Sheng H. Shao J. Williams C.S. Pisacane P.I. Sliwkowski M.X. DuBois R.N. Gastroenterology. 2001; 120: 1713-1719Google Scholar), combined therapy with a selective COX-2 inhibitor, celecoxib, and an antibody to Her-2/neu, a member of the ErbB/Her family of growth factor receptors, results in almost complete inhibition of the growth of xenografted colon cancers. The mechanisms by which these combinatorial treatments produce remarkable results were not clear, and choosing these therapeutic targets was based on the importance of individual signaling pathways. Our studies suggest that COX-2/PGE2 and the EGFR signaling system synergistically induce the expression of critical genes for colonic neoplasms and, therefore, provide a potential mechanism for the combinatorial treatment of colonic neoplasia targeting both the COX-2/PGE2 signaling pathway and the EGFR signaling system that has demonstrated significant advantages. We thank Dr. Sean B. Lee (NIDDK, National Institutes of Health) for providing amphiregulin promoter constructs and Dr. Michael White (University of Texas Southwestern Medical Center, Dallas, TX) for providing Raf expression construct." @default.
• W2024579029 created "2016-06-24" @default.
• W2024579029 creator A5005959278 @default.
• W2024579029 creator A5035855141 @default.
• W2024579029 creator A5066347342 @default.
• W2024579029 date "2004-04-01" @default.
• W2024579029 modified "2023-10-03" @default.
• W2024579029 title "Prostaglandin E2 Synergistically Enhances Receptor Tyrosine Kinase-dependent Signaling System in Colon Cancer Cells" @default.
• W2024579029 cites W1482218984 @default.
• W2024579029 cites W1494401713 @default.
• W2024579029 cites W1514189093 @default.
• W2024579029 cites W1606885912 @default.
• W2024579029 cites W1975131304 @default.
• W2024579029 cites W1979201752 @default.
• W2024579029 cites W1993163893 @default.
• W2024579029 cites W1993592556 @default.
• W2024579029 cites W2009160843 @default.
• W2024579029 cites W2010433707 @default.
• W2024579029 cites W2031844898 @default.
• W2024579029 cites W2045401053 @default.
• W2024579029 cites W2048680922 @default.
• W2024579029 cites W2050202520 @default.
• W2024579029 cites W2051686420 @default.
• W2024579029 cites W2060973430 @default.
• W2024579029 cites W2073738021 @default.
• W2024579029 cites W2075039358 @default.
• W2024579029 cites W2077668550 @default.
• W2024579029 cites W2085141824 @default.
• W2024579029 cites W2087744847 @default.
• W2024579029 cites W2088905899 @default.
• W2024579029 cites W2089449927 @default.
• W2024579029 cites W2093531978 @default.
• W2024579029 cites W2097997344 @default.
• W2024579029 cites W2106293055 @default.
• W2024579029 cites W2120226344 @default.
• W2024579029 cites W2120805579 @default.
• W2024579029 cites W2132707921 @default.
• W2024579029 cites W2138448256 @default.
• W2024579029 cites W2154608911 @default.
• W2024579029 cites W2156470937 @default.
• W2024579029 cites W2312652235 @default.
• W2024579029 cites W2318925269 @default.
• W2024579029 cites W2324578623 @default.
• W2024579029 cites W2331808804 @default.
• W2024579029 cites W2626800913 @default.
• W2024579029 cites W4211075026 @default.
• W2024579029 doi "https://doi.org/10.1074/jbc.m313276200" @default.
• W2024579029 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/14742435" @default.
• W2024579029 hasPublicationYear "2004" @default.
• W2024579029 type Work @default.
• W2024579029 sameAs 2024579029 @default.
• W2024579029 citedByCount "84" @default.
• W2024579029 countsByYear W20245790292012 @default.
• W2024579029 countsByYear W20245790292013 @default.
• W2024579029 countsByYear W20245790292014 @default.
• W2024579029 countsByYear W20245790292015 @default.
• W2024579029 countsByYear W20245790292016 @default.
• W2024579029 countsByYear W20245790292018 @default.
• W2024579029 countsByYear W20245790292020 @default.
• W2024579029 countsByYear W20245790292023 @default.
• W2024579029 crossrefType "journal-article" @default.
• W2024579029 hasAuthorship W2024579029A5005959278 @default.
• W2024579029 hasAuthorship W2024579029A5035855141 @default.
• W2024579029 hasAuthorship W2024579029A5066347342 @default.
• W2024579029 hasBestOaLocation W20245790291 @default.
• W2024579029 hasConcept C101544691 @default.
• W2024579029 hasConcept C121608353 @default.
• W2024579029 hasConcept C126322002 @default.
• W2024579029 hasConcept C134018914 @default.
• W2024579029 hasConcept C170493617 @default.
• W2024579029 hasConcept C178592051 @default.
• W2024579029 hasConcept C180361614 @default.
• W2024579029 hasConcept C185592680 @default.
• W2024579029 hasConcept C23440912 @default.
• W2024579029 hasConcept C2775960820 @default.
• W2024579029 hasConcept C2777956040 @default.
• W2024579029 hasConcept C2778938600 @default.
• W2024579029 hasConcept C42362537 @default.
• W2024579029 hasConcept C502942594 @default.
• W2024579029 hasConcept C526805850 @default.
• W2024579029 hasConcept C55493867 @default.
• W2024579029 hasConcept C62478195 @default.
• W2024579029 hasConcept C71924100 @default.
• W2024579029 hasConcept C79186569 @default.
• W2024579029 hasConcept C86803240 @default.
• W2024579029 hasConcept C95444343 @default.
• W2024579029 hasConceptScore W2024579029C101544691 @default.
• W2024579029 hasConceptScore W2024579029C121608353 @default.
• W2024579029 hasConceptScore W2024579029C126322002 @default.
• W2024579029 hasConceptScore W2024579029C134018914 @default.
• W2024579029 hasConceptScore W2024579029C170493617 @default.
• W2024579029 hasConceptScore W2024579029C178592051 @default.
• W2024579029 hasConceptScore W2024579029C180361614 @default.
• W2024579029 hasConceptScore W2024579029C185592680 @default.
• W2024579029 hasConceptScore W2024579029C23440912 @default.
• W2024579029 hasConceptScore W2024579029C2775960820 @default.
• W2024579029 hasConceptScore W2024579029C2777956040 @default.
• W2024579029 hasConceptScore W2024579029C2778938600 @default.

• next