Prostaglandin E2 Synergistically Enhances Receptor Tyrosine Kinase-dependent Signaling System in Colon Cancer Cells

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 gene using the and Renilla were using a assay system and a were to Renilla and as The expression the of kinase by The expression and expression were purchased from The expression and the CREB, were purchased analysis was as described previously J. Sheng H. H. Morrow J.D. DuBois R.N. J. Biol. Chem. 2000; 275: Scholar). Cells were for in assay 0.5% 1 and cell were and by After the proteins were to and the filters were with the antibodies indicated and developed by the system The and antibodies were purchased from Cell MA). of the were on a with the were by between were using the with a of were as between PGE2 and the Ras/Raf/MAPK growth and transformation the expression of a of pro-oncogenic plays critical roles in colon cancer cell proliferation and transformation A. C. A. A. S. P. F. N. Oncogene. 2000; Scholar, S.K. C.J. Coffey Jr., R.J. J. Cancer. 1999; Scholar). We have demonstrated that PGE2 and TGF-α 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 role in of signals from PGE2 and oncogenic induces AR transcription in a synergistic (8Shao J. Lee S.B. Guo H. Evers B.M. Sheng H. Cancer Res. 2003; 63: 5218-5223Google Scholar). the interaction between PGE2 and the Ras/Raf/MAPK pathway, we and MAPK/ERK kinase LS-174 cells. Transfection with an expression (16Sheng H. Shao H. DuBois R.N. J. Biol. Chem. 2001; 276: 14498-14504Google Scholar), increased AR transcription The of PGE2 in a in the activity of AR promoter when with the cells with In expression of a not AR transcription. expression of an of increased AR promoter activity a synergistic induction was between PGE2 and activity These results suggest a synergy between PGE2 signaling and the Ras/Raf/MAPK pathway in of AR transcription. TGF-α signals through the EGFR and RTK-dependent signaling PGE2 acts through induction of cAMP and activation of protein kinase A of EGFR tyrosine kinases or the pathway the synergy of TGF-α and a or a specific EGFR tyrosine kinase the synergistic of PGE2 and treatment with H-89 and PD-153035 blocked the induction of AR transcription by PGE2 and TGF-α of the Sp1 in AR AR promoter a number of functional as demonstrated in determine the of element in AR deletion constructs were The activity with pGL2-A, and pGL2-A2 was that with upper The promoter activity was to between and an Sp1 to was of the Sp1 AR promoter Luciferase activity was by in cells. The which the AR promoter from to including the a tumor element a CRE, and a was PGE2 and TGF-α synergistically induced the activity in LS-174 suggesting are for the PGE2 and TGF-α synergy on AR transcription of in the PGE2 and TGF-α 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). the role of the Sp1 and the in synergistic induction of AR the element was in pGL2-A2 and referred to as the basal activity of pGL2-A2 and pGL2-A2ΔCRE was PGE2 not the activity in LS-174 cells. The synergistic effect of PGE2 and TGF-α was by disruption of the Furthermore, induction of AR transcription by oncogenic required the The synergistic induction of AR transcription by oncogenic and PGE2 was when LS-174 cells were with pGL2-A2ΔCRE the critical role of the in AR we co-transfected LS-174 cells with a expression and of increased the of activity In expression of a of the basal AR transcription and significantly blocked PGE2 activation of the AR promoter as In with the of cAMP significantly increased AR promoter A synergistic induction of AR transcription was when LS-174 cells were with both cAMP and TGF-α The AR transcription was synergistically enhanced by cAMP treatment as further determine the critical role of the in of AR LS-174 cells were with which a tumor the CRE, and a of the Sp1 element significantly AR promoter The basal activity of was of the activity of the the of AR promoter activity was in LS-174 cells PGE2 induced activity and PGE2 and TGF-α synergistically increased activity of the the activity of the AR results were in LS-174 cells that were with promoter which to -192) including the and a We have demonstrated that PGE2 treatment induces the of at studies indicate that TGF-α may and activation of through the pathway D. S. A. P. Proc. Natl. Acad. Sci. U. S. A. 1998; Scholar, N. J. P. J. Biol. Chem. 1998; Scholar). analysis that TGF-α treatment increased of ERK, Rsk, and in LS-174 cells PGE2 and TGF-α Cancer Cell the EGFR signaling system and the COX-2/PGE2 signaling system can synergistically pro-oncogenic gene was of to determine signaling systems tumorigenic We elucidated the growth effects of PGE2 and TGF-α in colon cancer cells. LS-174 cells are to when are in (7Sheng H. Shao J. Washington M.K. DuBois R.N. J. Biol. Chem. 2001; 276: 18075-18081Google Scholar). The of of cells and a by LS-174 cells in were to LS-174 in mice The of TGF-α or PGE2 increased the of In TGF-α and PGE2 synergistically effects on LS-174 cells cancer T-84 which have been reported to to PGE2 treatment Y. K. J. 1998; Scholar), in The synergistic stimulation of TGF-α and PGE2 on cell growth was in T-84 cells as well These results suggest that TGF-α and PGE2 may the growth of colon in a synergistic manner. PGE2 and TGF-α Cancer Cell studies have demonstrated that PGE2 promotes migration and 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). the other EGFR signaling is critical for colon cancer cell migration Cell Res. 1999; Scholar). the of cell migration by PGE2 and TGF-α, LS-174 cells were to the chamber assay LS-174 cells on the a that were not by cells. LS-174 cells when both PGE2 and TGF-α were in the chamber not a PGE2 and TGF-α synergistically stimulated LS-174 cell migration and increased the number of cells that the results were in colon cancer T-84 cells cancers and a number of genetic for B. Cell. 1996; 87: Scholar). studies a reduction in the of colorectal cancer and colorectal in on a M.M. Cancer Res. Scholar, S.H. C.M. Med. Scholar, E. N. J. Med. Scholar). disruption of the COX-2/PGE2 pathway results in a 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, M. Takaku K. Sasaki N. Sugimoto Y. Ushikubi F. Narumiya S. Oshima M. Taketo M.M. Nat. Med. 2001; 7: 1048-1051Google Scholar). These indicate the critical role of COX-2/PGE2 in colon cancer and an is the molecular mechanism that mediates the pro-oncogenic of the COX-2/PGE2 signaling pathway in colorectal studies have demonstrated that PGE2 induces and of the EGFR through 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). of the EGFR is critical for PGE2-induced growth and of colon cancer cells. neoplasms are with of the EGFR signaling system. growth factor receptors including their are in colorectal neoplasms H. E. M. S. H. N. Res. Scholar, C. E. D. A. 1997; Scholar). of EGFR including TGF-α, and in colonic is well A. C. A. A. S. P. F. N. Oncogene. 2000; Scholar, H. E. M. S. H. N. Res. Scholar, N. C. E. M. F. J. Cancer. Scholar). of the EGFR by PGE2 may further the activity of the EGFR the of the EGFR signaling system. mechanisms to be for the of the key roles of COX-2/PGE2 in colorectal neoplasia that have been in studies and in 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, M. Takaku K. Sasaki N. Sugimoto Y. Ushikubi F. Narumiya S. Oshima M. Taketo M.M. Nat. Med. 2001; 7: 1048-1051Google Scholar, M.M. Cancer Res. Scholar, S.H. C.M. Med. Scholar, E. N. J. Med. Scholar). Our results 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 signaling systems synergistically induced the expression of AR, the that COX-2/PGE2 may the expression of pro-oncogenic which are by growth and and which are critical for transformation of intestinal Thus far, COX-2/PGE2 is not as an oncogenic signaling our results suggest that COX-2/PGE2 may the activity of oncogenic signaling systems and exert pro-oncogenic actions to colonic that COX-2 is a gene of oncogenic K. N. R. B. Cancer Res. 1996; Scholar, H. Shao J. P. DuBois R.N. Beauchamp R.D. J. Biol. Chem. 1998; Scholar, H. Shao J. DuBois R.N. Cancer Res. 2001; Scholar). of COX-2 enzyme activity the growth of of intestinal epithelial cells in suggesting the of COX-2 in transformation Shao J. Sheng H. Isakson Morrow J.D. Coffey R.J. DuBois R.N. Beauchamp R.D. Gastroenterology. 1997; Scholar). We have reported that activation of receptor J. Sheng H. DuBois R.N. Cancer Res. 2002; Scholar), which has been shown to colon cancer cells from B. Cell. 1999; 99: Scholar). In this study, we that PGE2 synergistically enhanced transcription of a pro-oncogenic gene, AR, of which and in colorectal are well A. C. A. A. S. P. F. N. Oncogene. 2000; Scholar, N. C. E. M. F. J. Cancer. Scholar). These suggest that may to transformation of intestinal epithelial cells through mechanisms. the critical role of in the to carcinoma of in the transformation of colonic epithelial our results to the pro-oncogenic roles of the signaling system in the genetic pathway of colorectal neoplasia. The kinase is a of R. G.J. Der C.J. Oncogene. 1998; 17: Scholar). Activation of is and for the activation of the and the transcription the transcription of by the element H. M. C. C. J. Scholar). The pathway plays an role in the of cell cell cell and cell transformation J. Cell. 2000; Scholar) and can be by a large number of growth and through or mechanisms C. A. Biol. Cell. 2001; Scholar). Our results a PGE2, and PGE2, and in of AR suggesting that PGE2 is to the pathway at and a variety of cellular The human AR promoter a number of functional 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, J.M. C.M. G.J. M. Cell. Biol. Scholar). We that the activation of the AR promoter was by a and a The Sp1 element to provide the activity of the AR the was responsible for the activation of the of the Sp1 AR promoter activity by its to PGE2 and TGF-α In disruption of the maintained the basal activity of AR promoter the AR transcription. Our suggest that the pathway PGE2 signaling and was required for PGE2 induction of AR transcription. the other the effect of TGF-α on AR transcription was TGF-α treatment in of ERK, Rsk, and CREB, suggesting that TGF-α may AR transcription via as well D. S. A. P. Proc. Natl. Acad. Sci. U. S. A. 1998; Scholar, N. J. P. J. Biol. Chem. 1998; Scholar). are required to determine the mechanism by which PGE2 and TGF-α synergistically AR transcription. of agents that genetic or in neoplasms may in treatment. C.J. E. B. A. M. P. C.M. Nat. Med. 2000; Scholar) that a of a and an EGFR kinase remarkable from intestinal neoplasia in mice. mice developed intestinal by and the number of by of the mice with agents developed at In M. Sheng H. Shao J. DuBois R.N. Gastroenterology. 2001; Scholar), combined with a COX-2 and an to a member of the family of growth factor results in of the growth of colon cancers. The mechanisms by which combinatorial remarkable results were not and was on the of signaling Our studies suggest that COX-2/PGE2 and the EGFR signaling system synergistically the expression of critical for colonic neoplasms 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 advantages. We B. Lee of for amphiregulin promoter constructs and White of for expression

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