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Medical Oncology

, 33:73 | Cite as

Epac1 knockdown inhibits the proliferation of ovarian cancer cells by inactivating AKT/Cyclin D1/CDK4 pathway in vitro and in vivo

  • Meng Gao
  • Yanyan Ma
  • Robert C. BastJr.
  • Yue Li
  • Lu Wan
  • Yanping Liu
  • Yingshuo Sun
  • Zhenghui Fang
  • Lining Zhang
  • Xiaoyan WangEmail author
  • Zengtao WeiEmail author
Original Paper

Abstract

Ovarian cancer is the leading cause of death among gynecological malignancies, and high grade serous ovarian carcinoma is the most common and most aggressive subtype. Recently, it was demonstrated that cAMP mediates protein kinase A-independent effects through Epac (exchange protein directly activated by cAMP) proteins. Epac proteins, including Epac1 and Epac2, are implicated in several diverse cellular responses, such as insulin secretion, exocytosis, cellular calcium handling and formation of cell–cell junctions. Several reports document that Epac1 could play vital roles in promoting proliferation, invasion and migration of some cancer cells. However, the expression levels and roles of Epac1 in ovarian cancer have not been investigated. In the present study, we detected the expression levels of Epac1 mRNA and protein in three kinds of ovarian cancer cells SKOV3, OVCAR3 and CAOV3. Furthermore, the effect of Epac1 knockdown on the proliferation and apoptosis of SKOV3 and OVCAR3 cells was evaluated in vitro and in vivo. The results showed that there was higher expression of Epac1 mRNA and protein in SKOV3 and OVCAR3 cells. Epac1 knockdown inhibited the proliferation of SKOV3 and OVCAR3 cells in vitro and in vivo. Decreased proliferation may be due to downregulation of Epac1-induced G1 phase arrest by inactivating the AKT/Cyclin D1/CDK4 pathway, but not to alterations in the MAPK pathway or to apoptosis. Taken together, our data provide new insight into the essential role of Epac1 in regulating growth of ovarian cancer cells and suggest that Epac1 might represent an attractive therapeutic target for treatment of ovarian cancer.

Keywords

Epac1 Ovarian cancer cells Proliferation Cell cycle Apoptosis 

Notes

Acknowledgments

This work was supported by the National Natural Science Foundation of China (81471437, 81172863) and Natural Science Foundation of Shandong (ZR2012HM091, ZR2013HM105).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

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References

  1. 1.
    Dancey J. Targeted therapies and clinical trials in ovarian cancer. Ann Oncol. 2013;24(Suppl 10):x59–63. doi: 10.1093/annonc/mdt473.CrossRefPubMedGoogle Scholar
  2. 2.
    Sundar S, Neal RD, Kehoe S. Diagnosis of ovarian cancer. BMJ. 2015;351:h4443. doi: 10.1136/bmj.h4443.CrossRefPubMedGoogle Scholar
  3. 3.
    Marquez RT, Baggerly KA, Patterson AP, Liu J, Broaddus R, Frumovitz M, et al. Patterns of gene expression in different histotypes of epithelial ovarian cancer correlate with those in normal fallopian tube, endometrium, and colon. Clin Cancer Res. 2005;11(17):6116–26. doi: 10.1158/1078-0432.CCR-04-2509.CrossRefPubMedGoogle Scholar
  4. 4.
    Heintz APM, Odicino F, Maisonneuve P, Quinn MA, Benedet JL, Creasman WT, et al. Carcinoma of the Ovary. Int J Gynecol Obstet. 2006;95:S161–92. doi: 10.1016/s0020-7292(06)60033-7.CrossRefGoogle Scholar
  5. 5.
    Clarke-Pearson DL. Clinical practice. Screening for ovarian cancer. N Engl J Med. 2009;361:170–7.CrossRefPubMedGoogle Scholar
  6. 6.
    Lopez J, Banerjee S, Kaye SB. New developments in the treatment of ovarian cancer—future perspectives. Ann Oncol. 2013;24(Suppl 10):x69–76. doi: 10.1093/annonc/mdt475.CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Mayr M, Montminy M. Transcriptional regulation by the phosphorylation-dependent factor CREB. Nat Rev Mol Cell Biol. 2001;2:599–609.CrossRefPubMedGoogle Scholar
  8. 8.
    Beavo JA, Brunton LL. Cyclic nucleotide research—still expanding after half a century. Nat Rev Mol Cell Biol. 2002;3:710–8.CrossRefPubMedGoogle Scholar
  9. 9.
    de Rooij J, Zwartkruis FJ, Verheijen MH, Cool RH, Nijman SM, Wittinghofer A, et al. Epac is a Rap1 guanine-nucleotide-exchange factor directly activated by cyclic AMP. Nature. 1998;396:474–7.CrossRefPubMedGoogle Scholar
  10. 10.
    Bos JL. Epac: a new cAMP target and new avenues in cAMP research. Nat Rev Mol Cell Biol. 2003;4:733–8.CrossRefPubMedGoogle Scholar
  11. 11.
    Bos JL. Epac proteins: multi-purpose cAMP targets. Trends Biochem Sci. 2006;31(12):680–6. doi: 10.1016/j.tibs.2006.10.002.CrossRefPubMedGoogle Scholar
  12. 12.
    Bryn T, Mahic M, Enserink JM, Schwede F, Aandahl EM, Tasken K. The cyclic AMP-Epac1-Rap1 pathway is dissociated from regulation of effector functions in monocytes but acquires immunoregulatory function in mature macrophages. J Immunol. 2006;176(12):7361–70. doi: 10.4049/jimmunol.176.12.7361.CrossRefPubMedGoogle Scholar
  13. 13.
    Tiwari S, Felekkis K, Moon EY, Flies A, Sherr DH, Lerner A. Among circulating hematopoietic cells, B-CLL uniquely expresses functional EPAC1, but EPAC1-mediated Rap1 activation does not account for PDE4 inhibitor-induced apoptosis. Blood. 2004;103(7):2661–7. doi: 10.1182/blood-2003-06-2154.CrossRefPubMedGoogle Scholar
  14. 14.
    Lorenowicz MJ, van Gils J, de Boer M, Hordijk PL, Fernandez-Borja M. Epac1-Rap1 signaling regulates monocyte adhesion and chemotaxis. J Leukoc Biol. 2006;80(6):1542–52. doi: 10.1189/jlb.0506357.CrossRefPubMedGoogle Scholar
  15. 15.
    Kawasaki H, Springett GM, Mochizuki N, Toki S, Nakaya M, Matsuda M, et al. A family of cAMP-binding proteins that directly activate Rap1. Science. 1998;282:2275–9.CrossRefPubMedGoogle Scholar
  16. 16.
    Holz GG, Kang G, Harbeck M, Roe MW, Chepurny OG. Cell physiology of cAMP sensor Epac. J Physiol. 2006;577(Pt 1):5–15. doi: 10.1113/jphysiol.2006.119644.CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Kang G, Joseph JW, Chepurny OG, Monaco M, Wheeler MB, Bos JL, et al. Epac-selective cAMP analog 8-pCPT-2′-O-Me-cAMP as a stimulus for Ca2+-induced Ca2+ release and exocytosis in pancreatic beta-cells. J Biol Chem. 2003;278(10):8279–85. doi: 10.1074/jbc.M211682200.CrossRefPubMedGoogle Scholar
  18. 18.
    Ozaki N, Shibasaki T, Kashima Y, Miki T, Takahashi K, Ueno H, et al. cAMP-GEFII is a direct target of cAMP in regulated exocytosis. Nat Cell Biol. 2000;2:805–11.CrossRefPubMedGoogle Scholar
  19. 19.
    Oestreich EA, Wang H, Malik S, Kaproth-Joslin KA, Blaxall BC, Kelley GG, et al. Epac-mediated activation of phospholipase C(epsilon) plays a critical role in beta-adrenergic receptor-dependent enhancement of Ca2+ mobilization in cardiac myocytes. J Biol Chem. 2007;282(8):5488–95. doi: 10.1074/jbc.M608495200.CrossRefPubMedGoogle Scholar
  20. 20.
    Birukova AA, Zagranichnaya T, Fu P, Alekseeva E, Chen W, Jacobson JR, et al. Prostaglandins PGE(2) and PGI(2) promote endothelial barrier enhancement via PKA- and Epac1/Rap1-dependent Rac activation. Exp Cell Res. 2007;313:2504–20.CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Cullere X, Shaw SK, Andersson L, Hirahashi J, Luscinskas FW, Mayadas TN. Regulation of vascular endothelial barrier function by Epac, a cAMP-activated exchange factor for Rap GTPase. Blood. 2005;105(5):1950–5. doi: 10.1182/blood-2004-05-1987.CrossRefPubMedGoogle Scholar
  22. 22.
    Misra UK, Pizzo SV. Epac1-induced cellular proliferation in prostate cancer cells is mediated by B-Raf/ERK and mTOR signaling cascades. J Cell Biochem. 2009;108(4):998–1011. doi: 10.1002/jcb.22333.CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Almahariq M, Tsalkova T, Mei FC, Chen H, Zhou J, Sastry SK, et al. A novel EPAC-specific inhibitor suppresses pancreatic cancer cell migration and invasion. Mol Pharmacol. 2013;83(1):122–8. doi: 10.1124/mol.112.080689.CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Baljinnyam E, Umemura M, De Lorenzo MS, Iwatsubo M, Chen S, Goydos JS, et al. Epac1 promotes melanoma metastasis via modification of heparan sulfate. Pigment Cell Melanoma Res. 2011;24(4):680–7. doi: 10.1111/j.1755-148X.2011.00863.x.CrossRefPubMedGoogle Scholar
  25. 25.
    Qi J, Zhao P, Li F, Guo Y, Cui H, Liu A, et al. Down-regulation of Rab17 promotes tumourigenic properties of hepatocellular carcinoma cells via Erk pathway. Int J Clin Exp Pathol. 2015;8:4963–71.PubMedPubMedCentralGoogle Scholar
  26. 26.
    Rangarajan S, Enserink JM, Kuiperij HB, de Rooij J, Price LS, Schwede F, et al. Cyclic AMP induces integrin-mediated cell adhesion through Epac and Rap1 upon stimulation of the beta 2-adrenergic receptor. J Cell Biol. 2003;160(4):487–93. doi: 10.1083/jcb.200209105.CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Grandoch M, Rose A, ter Braak M, Jendrossek V, Rubben H, Fischer JW, et al. Epac inhibits migration and proliferation of human prostate carcinoma cells. Br J Cancer. 2009;101(12):2038–42. doi: 10.1038/sj.bjc.6605439.CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Yang L, Zhang HW, Hu R, Yang Y, Qi Q, Lu N, et al. Wogonin induces G1 phase arrest through inhibiting Cdk4 and cyclin D1 concomitant with an elevation in p21Cip1 in human cervical carcinoma HeLa cells. Biochem Cell Biol. 2009;87(6):933–42. doi: 10.1139/o09-060.CrossRefPubMedGoogle Scholar
  29. 29.
    Kim TH, Oh S, Kim SS. Recombinant human prothrombin kringle-2 induces bovine capillary endothelial cell cycle arrest at G0–G1 phase through inhibition of cyclin D1/CDK4 complex: modulation of reactive oxygen species generation and up-regulation of cyclin-dependent kinase inhibitors. Angiogenesis. 2005;8(4):307–14. doi: 10.1007/s10456-005-9020-y.CrossRefPubMedGoogle Scholar
  30. 30.
    Li T, Zhao X, Mo Z, Huang W, Yan H, Ling Z, et al. Formononetin promotes cell cycle arrest via downregulation of Akt/Cyclin D1/CDK4 in human prostate cancer cells. Cell Physiol Biochem. 2014;34(4):1351–8. doi: 10.1159/000366342.CrossRefPubMedGoogle Scholar
  31. 31.
    Shimura T, Noma N, Oikawa T, Ochiai Y, Kakuda S, Kuwahara Y, et al. Activation of the AKT/cyclin D1/Cdk4 survival signaling pathway in radioresistant cancer stem cells. Oncogenesis. 2012;1:e12. doi: 10.1038/oncsis.2012.12.CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Moon EY, Lee GH, Lee MS, Kim HM, Lee JW. Phosphodiesterase inhibitors control A172 human glioblastoma cell death through cAMP-mediated activation of protein kinase A and Epac1/Rap1 pathways. Life Sci. 2012;90(9–10):373–80. doi: 10.1016/j.lfs.2011.12.010.CrossRefPubMedGoogle Scholar
  33. 33.
    Sun L, Kondeti VK, Xie P, Raparia K, Kanwar YS. Epac1-mediated, high glucose-induced renal proximal tubular cells hypertrophy via the Akt/p21 pathway. Am J Pathol. 2011;179(4):1706–18. doi: 10.1016/j.ajpath.2011.06.035.CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Kwak HJ, Park KM, Choi HE, Chung KS, Lim HJ, Park HY. PDE4 inhibitor, roflumilast protects cardiomyocytes against NO-induced apoptosis via activation of PKA and Epac dual pathways. Cell Signal. 2008;20(5):803–14. doi: 10.1016/j.cellsig.2007.12.011.CrossRefPubMedGoogle Scholar
  35. 35.
    Qin Y, Stokman G, Yan K, Ramaiahgari S, Verbeek F, de Graauw M, et al. cAMP signalling protects proximal tubular epithelial cells from cisplatin-induced apoptosis via activation of Epac. Br J Pharmacol. 2012;165(4b):1137–50. doi: 10.1111/j.1476-5381.2011.01594.x.CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Chen C, Du J, Feng W, Song Y, Lu Z, Xu M, et al. Beta-adrenergic receptors stimulate interleukin-6 production through Epac-dependent activation of PKCdelta/p38 MAPK signalling in neonatal mouse cardiac fibroblasts. Br J Pharmacol. 2012;166(2):676–88. doi: 10.1111/j.1476-5381.2011.01785.x.CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Meng Gao
    • 1
  • Yanyan Ma
    • 1
  • Robert C. BastJr.
    • 2
  • Yue Li
    • 1
  • Lu Wan
    • 1
  • Yanping Liu
    • 3
    • 4
  • Yingshuo Sun
    • 3
    • 4
  • Zhenghui Fang
    • 3
  • Lining Zhang
    • 1
  • Xiaoyan Wang
    • 1
    Email author
  • Zengtao Wei
    • 3
    • 4
    Email author
  1. 1.Department of ImmunologyShandong University School of MedicineJinanPeople’s Republic of China
  2. 2.Department of Experimental Therapeutics, Division of Cancer MedicineThe University of Texas MD Anderson Cancer CenterHoustonUSA
  3. 3.Department of Gynecology and ObstetricsJinan Central Hospital Affiliated to Shandong UniversityJinanPeople’s Republic of China
  4. 4.Department of Gynecology and ObstetricsShandong University School of MedicineJinanPeople’s Republic of China

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