Targeting mTOR and eIF4E: a feasible scenario in ovarian cancer therapy

Abstract

Ovarian carcinoma is one of the most common causes for cancer death in women; lack of early diagnosis and acquired resistance to platinum-based chemotherapy account for its poor prognosis and high mortality rate. As with other cancer types, ovarian cancer is characterized by dysregulated signaling pathways and protein synthesis, which together contribute to rapid cellular growth and invasiveness. The mechanistic/mammalian target of rapamycin (mTOR) pathway represents the core of different signaling pathways regulating a number of essential steps in the cell, among which protein synthesis and the eukaryotic initiation factor 4E (eIF4E), the mRNA cap binding protein, is one of its downstream effectors. eIF4E is a limiting factor in translation initiation and its overexpression is a hallmark in many cancers. Because its action is regulated by a number of factors that compete for the same binding site, eIF4E is an ideal target for developing novel antineoplastic drugs. Several inhibitors targeting the mTOR signaling pathway have been designed thus far, however most of these molecules show poor stability and high toxicity in vivo. This minireview explores the possibility of targeting mTOR and eIF4E proteins, thus impacting on translation initiation in ovarian cancer, describing the most promising experimental strategies and specific inhibitors that have been shown to have an effect on other kinds of cancers.

Keywords

Ovarian cancer / targeted therapy / mTOR pathway / eIF4E / inhibitors

References

[1] Siegel RL,Jemal A.Cancer statistics, 2016.CA Cancer J Clin2016;66:7-30

[2] American Cancer Society, 2021 Available from: https://www.cancer.org/cancer/ovarian-cancer/about/key-statistics.html [Last accessed on 6 May 2021]

[3] Yurkovetsky Z,Lomakin A.Development of a multimarker assay for early detection of ovarian cancer.J Clin Oncol2010;28:2159-66 PMCID:PMC2860434

[4] Torre LA,DeSantis CE.Ovarian cancer statistics, 2018.CA Cancer J Clin2018;68:284-96 PMCID:PMC6621554

[5] Lloyd KL,Savage RS.Prediction of resistance to chemotherapy in ovarian cancer: a systematic review.BMC Cancer2015;15:117 PMCID:PMC4371880

[6] Momenimovahed Z,Taheri S.Ovarian cancer in the world: epidemiology and risk factors.Int J Womens Health2019;11:287-99 PMCID:PMC6500433

[7] Helm CW.Enhancing the efficacy of cisplatin in ovarian cancer treatment - could arsenic have a role.J Ovarian Res2009;2:2 PMCID:PMC2636805

[8] Agarwal R.Ovarian cancer: strategies for overcoming resistance to chemotherapy.Nat Rev Cancer2003;3:502-16

[9] Metzger-Filho O,D'Hondt V.First-line systemic treatment of ovarian cancer: a critical review of available evidence and expectations for future directions.Curr Opin Oncol2010;22:513-20

[10] Armstrong DK.Relapsed ovarian cancer: challenges and management strategies for a chronic disease.Oncologist2002;7 Suppl 5:20-8

[11] Bast RC Jr,Mills GB.The biology of ovarian cancer : new opportunities for translation.Nat Rev Cancer2009;9:415-28 PMCID:PMC2814299

[12] Eckstein N.Platinum resistance in breast and ovarian cancer cell lines.J Exp Clin Cancer Res2011;30:91 PMCID:PMC3197542

[13] Damia G.Platinum Resistance in Ovarian Cancer: Role of DNA Repair.Cancers (Basel)2019;11:119 PMCID:PMC6357127

[14] El-Shaimaa A.Arafa, Zhu Q, et al. Tangeretin Sensitizes Cisplatin-resistant Human Ovarian Cancer Cells through Down-regulation of PI3K/Akt Signaling Pathway.Cancer Res2009;23:8910-7. PMCID:PMC3319094

[15] Chen SH.New Insights into Mechanisms of Cisplatin Resistance: From Tumor Cell to Microenvironment.Int J Mol Sci2019;20:4136 PMCID:PMC6747329

[16] Xing D.Modeling Resistance to Pathway-Targeted Therapy in Ovarian Cancer.Cell Cycle2005;4:1004-6 PMCID:PMC2268865

[17] Rottenberg S,Perego P.The rediscovery of platinum-based cancer therapy.Nat Rev Cancer2021;21:37-50

[18] Beale PJ,Boxall F,Kelland LR.BCL-2 family protein expression and platinum drug resistance in ovarian carcinoma.Br J Cancer2000;82:436-40 PMCID:PMC2363292

[19] Bieg D,Nowak E.Morin decreases galectin-3 expression and sensitizes ovarian cancer cells to cisplatin.Arch Gynecol Obstet2018;298:1181-94 PMCID:PMC6244704

[20] Mansouri A,Ridgway LD,Claret FX.Cisplatin resistance in an ovarian carcinoma is associated with a defect in programmed cell death control through XIAP regulation.Oncol Res2003;13:399-404 PMCID:PMC4089892

[21] Li J,Kim JM.Human ovarian cancer and cisplatin resistance: possible role of inhibitor of apoptosis proteins.Endocrinology2001;142:370-80

[22] Gordon AN,Guthrie D,Gore ME.Recurrent epithelial ovarian carcinoma: a randomized phase III study of pegylated liposomal doxorubicin versus topotecan.J Clin Oncol2001;19:3312-22

[23] Mutch DG,Goss T.Randomized phase III trial of gemcitabine compared with pegylated liposomal doxorubicin in patients with platinum-resistant ovarian cancer.J Clin Oncol2007;25:2811-8

[24] Gogineni V,Staats H.Current Ovarian Cancer Maintenance Strategies and Promising New Developments.J Cancer2021;12:38-53 PMCID:PMC7738841

[25] Cortez AJ,Kujawa KA.Advances in ovarian cancer therapy.Cancer Chemother Pharmacol2018;81:17-38 PMCID:PMC5754410

[26] Tahmasebi S,Hershey JWB.Protein Synthesis and Translational Control: A Historical Perspective.Cold Spring Harb Perspect Biol2019;11:a035584 PMCID:PMC6719597

[27] Grzmil M.Translation regulation as a therapeutic target in cancer.Cancer Res2012;72:3891-900

[28] Liu GY.mTOR at the nexus of nutrition, growth, ageing and disease.Nat Rev Mol Cell Biol2020;21:183-203 PMCID:PMC7102936

[29] Laplante M.mTOR signaling in growth control and disease.Cell2012;149:274-93 PMCID:PMC3331679

[30] Grabiner BC,Birsoy K.A diverse array of cancer-associated mTOR mutations are hyperactivating and can predict rapamycin sensitivity.Cancer Discov2014;4:554-63 PMCID:PMC4012430

[31] Sabatini DM.mTOR and cancer: insights into a complex relationship.Nat Rev Cancer2006;6:729-34

[32] Fruman DA.PI3K and Cancer: Lessons, Challenges and Opportunities.Nat Rev Drug Discov2014;13:140-56 PMCID:PMC3994981

[33] Cossa G,Cassinelli G.Differential outcome of MEK1/2 inhibitor-platinum combinations in platinum-sensitive and -resistant ovarian carcinoma cells.Cancer Lett2014;347:212-24

[34] Deng J,Feng X.Inhibition of PI3K/Akt/mTOR signaling pathway alleviates ovarian cancer chemoresistance through reversing epithelial-mesenchymal transition and decreasing cancer stem cell marker expression.BMC Cancer2019;19:618 PMCID:PMC6591840

[35] Mabuchi S,Takahashi R.The PI3K/AKT/mTOR pathway as a therapeutic target in ovarian cancer.Gynecol Oncol2015;137:173-9

[36] Mabuchi S,Kimura T.Targeting mTOR signaling pathway in ovarian cancer.Curr Med Chem2011;18:2960-8

[37] Siddiqui N.Signalling to eIF4E in cancer.Biochem Soc Trans2015;43:763-72 PMCID:PMC4613458

[38] Hay N.Upstream and downstream of mTOR.Genes Dev2004;18:1926-45

[39] Bhat M,Hulea L,Pelletier J.Targeting the translation machinery in cancer.Nat Rev Drug Discov2015;14:261-78

[40] Wendel HG,Malina A.Dissecting eIF4E action in tumorigenesis.Genes Dev2007;21:3232-7 PMCID:PMC2113024

[41] Zinzalla V,Oppliger W.Activation of mTORC2 by association with the ribosome.Cell2011;144:757-68

[42] Noske A,Darb-esfahani S.Activation of mTOR in a subgroup of ovarian carcinomas : Correlation with p-eIF-4E and prognosis.Oncol Rep2008;20:1409-17

[43] Lu C,Wu D.Targeting translation: eIF4E as an emerging anticancer drug target.Expert Rev Mol Med2016;18:e2

[44] Li J,Blenis J.Rapamycin: one drug, many effects.Cell Metab2014;19:373-9 PMCID:PMC3972801

[45] Hudes G,Tomczak P.Global ARCC TrialTemsirolimus, interferon alfa, or both for advanced renal-cell carcinoma.N Engl J Med2007;356:2271-81

[46] Rini BI.Temsirolimus, an inhibitor of mammalian target of rapamycin.Clin Cancer Res2008;14:1286-90

[47] Houghton PJ.Everolimus.Clin Cancer Res2010;16:1368-72 PMCID:PMC3003868

[48] Mabuchi S,Connolly DC.RAD001 (Everolimus) delays tumor onset and progression in a transgenic mouse model of ovarian cancer.Cancer Res2007;67:2408-13

[49] Mita M,Abdel-Karim I,Giles F.Deforolimus (AP23573) a novel mTOR inhibitor in clinical development.Expert Opin Investig Drugs2008;17:1947-54

[50] Pópulo H,Soares P.The mTOR signalling pathway in human cancer.Int J Mol Sci2012;13:1886-918 PMCID:PMC3291999

[51] Pon YL,Cheung AN,Wong AS.p70 S6 kinase promotes epithelial to mesenchymal transition through snail induction in ovarian cancer cells.Cancer Res2008;68:6524-32

[52] Kurgan N,Kouvelioti R,Klentrou P.Inhibition of Human Lung Cancer Cell Proliferation and Survival by Post-Exercise Serum Is Associated with the Inhibition of Akt, mTOR, p70 S6K, and Erk1/2.Cancers (Basel)2017;9:46 PMCID:PMC5447956

[53] Ip CK.Exploiting p70 S6 kinase as a target for ovarian cancer.Expert Opin Ther Targets2012;16:619-30

[54] Lamming DW.Inhibition of the Mechanistic Target of Rapamycin (mTOR)-Rapamycin and Beyond.Cold Spring Harb Perspect Med2016;6:a025924 PMCID:PMC4852795

[55] Thoreen CC.Rapamycin inhibits mTORC1, but not completely.Autophagy2009;5:725-6

[56] Hsieh AC,Edlind MP.The translational landscape of mTOR signalling steers cancer initiation and metastasis.Nature2013;485:55-61 PMCID:PMC3663483

[57] Hsieh AC,Edlind MP.The translational landscape of mTOR signalling steers cancer initiation and metastasis.Nature2012;485:55-61

[58] Wong Te Fong AC,Gagrica S.Evaluation of the combination of the dual m-TORC1/2 inhibitor vistusertib (AZD2014) and paclitaxel in ovarian cancer models.Oncotarget2017;8:113874-84 PMCID:PMC5768370

[59] Zou ZQ,Wang F.A novel dual PI3Kalpha/mTOR inhibitor PI-103 with high antitumor activity in non-small cell lung cancer cells.Int J Mol Med2009;24:97-101

[60] Maira SM,Brueggen J.Identification and characterization of NVP-BEZ235, a new orally available dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor with potent in vivo antitumor activity.Mol Cancer Ther2008;7:1851-63

[61] Garlich JR,Dey N.A vascular targeted pan phosphoinositide 3-kinase inhibitor prodrug, SF1126, with antitumor and antiangiogenic activity.Cancer Res2008;68:206-15

[62] Heffron TP,Castanedo G.Identification of GNE-477, a potent and efficacious dual PI3K/mTOR inhibitor.Bioorg Med Chem Lett2010;20:2408-11

[63] Yu K,Shi C.Biochemical, cellular, and in vivo activity of novel ATP-competitive and selective inhibitors of the mammalian target of rapamycin.Cancer Res2009;69:6232-40

[64] Duffy AG,Ulahannan SV.Modulation of tumor eIF4E by antisense inhibition: A phase I/II translational clinical trial of ISIS 183750-an antisense oligonucleotide against eIF4E-in combination with irinotecan in solid tumors and irinotecan-refractory colorectal cancer.Int J Cancer2016;139:1648-57 PMCID:PMC6407898

[65] Hong DS,Oh Y.A phase 1 dose escalation, pharmacokinetic, and pharmacodynamic evaluation of eIF-4E antisense oligonucleotide LY2275796 in patients with advanced cancer.Clin Cancer Res2011;17:6582-91 PMCID:PMC5036398

[66] Jiang CC,Tseng HY.Repression of microRNA-768-3p by MEK/ERK signalling contributes to enhanced mRNA translation in human melanoma.Oncogene2014;33:2577-88

[67] Liu S,Lei M.Inhibiting ERK/Mnk/eIF4E broadly sensitizes ovarian cancer response to chemotherapy.Clin Transl Oncol2018;20:374-81

[68] Diab S,Yu M.MAP kinase-interacting kinases--emerging targets against cancer.Chem Biol2014;21:441-52

[69] Altman JK,Konicek BW.Inhibition of Mnk kinase activity by cercosporamide and suppressive effects on acute myeloid leukemia precursors.Blood2013;121:3675-81 PMCID:PMC3643766

[70] Hofmann WP,Sarrazin C.Ribavirin mode of action in chronic hepatitis C: from clinical use back to molecular mechanisms.Liver Int2008;28:1332-43

[71] Assouline S,Cocolakis E.Molecular targeting of the oncogene eIF4E in acute myeloid leukemia (AML): a proof-of-principle clinical trial with ribavirin.Blood2009;114:257-60

[72] Borden KL.Ribavirin as an anti-cancer therapy: acute myeloid leukemia and beyond?.Leuk Lymphoma2010;51:1805-15 PMCID:PMC2950216

[73] Casaos J,Huq S.The Use of Ribavirin as an Anticancer Therapeutic: Will It Go Viral?.Mol Cancer Ther2019;18:1185-94

[74] Jin J,Wu S,Xiao M.Targeting eIF4E signaling with ribavirin as a sensitizing strategy for ovarian cancer.Biochem Biophys Res Commun2019;510:580-6

[75] Dai D,Tang J.Inhibition of mTOR/eIF4E by anti-viral drug ribavirin effectively enhances the effects of paclitaxel in oral tongue squamous cell carcinoma.Biochem Biophys Res Commun2017;482:1259-64

[76] Kentsis A,Culjkovic B,Borden KL.Ribavirin suppresses eIF4E-mediated oncogenic transformation by physical mimicry of the 7-methyl guanosine mRNA cap.Proc Natl Acad Sci U S A2004;101:18105-10

[77] Yan Y,Lee JM,Pelletier J.Ribavirin is not a functional mimic of the 7-methyl guanosine mRNA cap.RNA2005;11:1238-44

[78] Westman B,Grudzien E.The antiviral drug ribavirin does not mimic the 7-methylguanosine moiety of the mRNA cap structure in vitro.RNA2005;11:1505-13

[79] Chen EZ,Patel MR.Small-molecule inhibition of oncogenic eukaryotic protein translation in mesothelioma cells.Invest New Drugs2014;32:598-603

[80] Li S,Jacobson B.Treatment of breast and lung cancer cells with a N-7 benzyl guanosine monophosphate tryptamine phosphoramidate pronucleotide (4Ei-1) results in chemosensitization to gemcitabine and induced eIF4E proteasomal degradation.Mol Pharm2013;10:523-31 PMCID:PMC5920544

[81] Moerke NJ,Chen H.Small-molecule inhibition of the interaction between the translation initiation factors eIF4E and eIF4G.Cell2007;128:257-67

[82] Papadopoulos E,Kabha E.Structure of the eukaryotic translation initiation factor eIF4E in complex with 4EGI-1 reveals an allosteric mechanism for dissociating eIF4G.Proc Natl Acad Sci U S A2014;111:E3187-95 PMCID:PMC4128100

[83] Cencic R,Robert F.Reversing chemoresistance by small molecule inhibition of the translation initiation complex eIF4F.Proc Natl Acad Sci U S A2011;108:6689 PMCID:PMC3024666

[84] Ko SY,Barengo N.Inhibition of ovarian cancer growth by a tumor-targeting peptide that binds eukaryotic translation initiation factor 4E.Clin Cancer Res2009;15:4336-47

[85] Lama D,Verma CS.Rational optimization of conformational effects induced by hydrocarbon staples in peptides and their binding interfaces.Sci Rep2013;3:3451 PMCID:PMC6506440

[86] Lama D,Brown CJ,Verma CS.159 Stapled-peptides targeting the protein-binding interface of eukaryotic Translation Initiation Factor 4E (eIF4E) protein.J Biomol Struct Dyn2015;33:102-3

[87] Lama D,Frosi Y.Structural insights reveal a recognition feature for tailoring hydrocarbon stapled-peptides against the eukaryotic translation initiation factor 4E protein.Chem Sci2019;10:2489-500 PMCID:PMC6385854

[88] Mazzoletti M.PI3K/AKT/mTOR inhibitors in ovarian cancer.Curr Med Chem2010;17:4433-47

[89] Langcake P.The production of resveratrol and the viniferins by grapevines in response to ultraviolet irradiation.Phytochemistry1977;16:1193-6

[90] Jang M,Udeani GO.Cancer chemopreventive activity of resveratrol, a natural product derived from grapes.Science1997;275:218-20

[91] Cao Z,Xia C,Jiang BH.trans-3,4,5'-Trihydroxystibene inhibits hypoxia-inducible factor 1alpha and vascular endothelial growth factor expression in human ovarian cancer cells.Clin Cancer Res2004;10:5253-63

[92] Zhong H,Laughner E.Overexpression of Hypoxia-inducible Factor 1α in Common Human Cancers and Their Metastase.Cancer Res2000;59:5830-5

[93] Zhong LX,Wu ML.Resveratrol and STAT inhibitor enhance autophagy in ovarian cancer cells.Cell Death Discov2016;2:15071 PMCID:PMC4979504

[94] Stakleff KS,Blanco D,Booth TD.Resveratrol exerts differential effects in vitro and in vivo against ovarian cancer cells.Asian Pac J Cancer Prev2012;13:1333-40

[95] Altman MK,Jia W.Suppression of the GTPase-activating protein RGS10 increases Rheb-GTP and mTOR signaling in ovarian cancer cells.Cancer Lett2015;369:175-83. PMCID:PMC4654121

[96] Lheureux S,Briand M.(18)F-FDG Is a Surrogate Marker of Therapy Response and Tumor Recovery after Drug Withdrawal during Treatment with a Dual PI3K/mTOR Inhibitor in a Preclinical Model of Cisplatin-Resistant Ovarian Cancer.Transl Oncol2013;6:586-95 PMCID:PMC3799200

[97] Fabi F,Parent S,Cadrin M.Pharmacologic inhibition of Akt in combination with chemotherapeutic agents effectively induces apoptosis in ovarian and endometrial cancer cell lines.Mol Oncol2020;

[98] Hu L,Lu Y,Jaffe RB.Inhibition of phosphatidylinositol 3’-kinase increases efficacy of paclitaxel in in vitro and in vivo ovarian cancer models. Cancer Res 2002;62:1087-92.

[99] David-West G,Gadi A.mTORC1/2 inhibition re-sensitizes platinum-resistant ovarian cancer by disrupting selective translation of DNA damage and survival mRNAs.Oncotarget2018;9:33064-76 PMCID:PMC6145695

[100] Jacobson BA,Kratzke MG.Repression of cap-dependent translation attenuates the transformed phenotype in non-small cell lung cancer both in vitro and in vivo.Cancer Res2006;66:4256-62

[101] Pelletier J,Ruggero D.Targeting the eIF4F translation initiation complex: a critical nexus for cancer development.Cancer Res2015;75:250-63 PMCID:PMC4299928

[102] Graff JR.Translational control and metastatic progression: Enhanced activity of the mRNA cap-binding protein eIF-4E selectively enhances translation of metastasis-related mRNAs.Clin Exp Metastasis2003;20:265-73

[103] Pettersson F,Emond A.Genetic and pharmacologic inhibition of eIF4E reduces breast cancer cell migration, invasion, and metastasis.Cancer Res2015;75:1102-12

[104] Asimomytis A,Rodolakis A.mTOR downstream effectors, 4EBP1 and eIF4E, are overexpressed and associated with HPV status in precancerous lesions and carcinomas of the uterine cervix.Oncol Lett2016;12:3234-40 PMCID:PMC5103924

[105] Yang S,Steinberg S,Swain S.Expression levels of eIF4E, VEGF, and cyclin D1, and correlation of eIF4E with VEGF and cyclin D1 in multi-tumor tissue microarray.Oncol Rep2007;17:281-7

[106] Zheng J,Zhang C.eIF4E Overexpression Is Associated with Poor Prognoses of Ovarian Cancer.Anal Cell Pathol (Amst)2020;2020:8984526 PMCID:PMC7787841

[107] Ueda T,Fukuyama H,Fukunaga R.Mnk2 and Mnk1 are essential for constitutive and inducible phosphorylation of eukaryotic initiation factor 4E but not for cell growth or development.Mol Cell Biol2004;24:6539-49 PMCID:PMC444855

[108] Waskiewicz AJ,Penn B,Kimball SR.Phosphorylation of the cap-binding protein eukaryotic translation initiation factor 4E by protein kinase Mnk1 in vivo.Mol Cell Biol1999;19:1871-80 PMCID:PMC83980

[109] McKendrick L,Pain VM,Joshi B.Phosphorylation of eukaryotic initiation factor 4E (eIF4E) at Ser209 is not required for protein synthesis in vitro and in vivo.Eur J Biochem2001;268:5375-85

[110] Carter JH,Spaulding NR IV.Phosphorylation of eIF4E serine 209 is associated with tumour progression and reduced survival in malignant melanoma.Br J Cancer2016;114:444-53 PMCID:PMC4815768

[111] Schneider RJ.Translational Control in Cancer Development and Progression.Cold Spring Harb Monogr Arch2007;48:401

[112] Wan J,Xu Z.Knockdown of eIF4E suppresses cell proliferation, invasion and enhances cisplatin cytotoxicity in human ovarian cancer cells.Int J Oncol2015;47:2217-25

[113] Zhan Y,Rajakumar A.The role of eIF4E in response and acquired resistance to vemurafenib in melanoma.J Invest Dermatol2015;135:1368-76

[114] Zhou FF,Guo GF.Knockdown of eIF4E suppresses cell growth and migration, enhances chemosensitivity and correlates with increase in Bax/Bcl-2 ratio in triple-negative breast cancer cells.Med Oncol2011;28:1302-7

[115] Choi CH,Kim SR.Direct inhibition of eIF4E reduced cell growth in endometrial adenocarcinoma.J Cancer Res Clin Oncol2011;137:463-9

[116] Cao J,Zhang X.Inhibition of eIF4E cooperates with chemotherapy and immunotherapy in renal cell carcinoma.Clin Transl Oncol2018;20:761-7

[117] Pettersson F,Dobocan MC.Ribavirin treatment effects on breast cancers overexpressing eIF4E, a biomarker with prognostic specificity for luminal B-type breast cancer.Clin Cancer Res2011;17:2874-84 PMCID:PMC3086959

[118] Jia Y,Amin EA,Bitterman PB.Design, synthesis and evaluation of analogs of initiation factor 4E (eIF4E) cap-binding antagonist Bn7-GMP.Eur J Med Chem2010;45:1304-13 PMCID:PMC2853597

[119] Ghosh B,Ghosh P.Nontoxic chemical interdiction of the epithelial-to-mesenchymal transition by targeting cap-dependent translation.ACS Chem Biol2009;4:367-77 PMCID:PMC2796976

[120] Sekiyama N,Papadopoulos E,Wagner G.Molecular mechanism of the dual activity of 4EGI-1: Dissociating eIF4G from eIF4E but stabilizing the binding of unphosphorylated 4E-BP1.Proc Natl Acad Sci U S A2015;112:E4036-45 PMCID:PMC4522750

[121] Marino D, Chillemi G, De Rubeis S, Tramontano A, Achsel T, Bagni C. MD and Docking Studies Reveal That the Functional Switch of CYFIP1 is Mediated by a Butterfly-like Motion.J Chem Theory Comput2015;11:3401-10

[122] Marino D, D'Annessa I, Tancredi H, Bagni C, Gallicchio E. A unique binding mode of the eukaryotic translation initiation factor 4E for guiding the design of novel peptide inhibitors.Protein Sci2015;24:1370-82 PMCID:PMC4570532