An overview of resistance to Human epidermal growth factor receptor 2 (Her2) targeted therapies in breast cancer

Abstract

Breast cancer (BC) is the second most common cause of cancer-related deaths and the most frequently diagnosed cancer in females. Among breast cancer types, HER2-positive breast cancer occurs in nearly 20% of human breast cancers and is associated with increased aggressiveness, poor prognosis, and shortened overall survival. HER2+ breast cancer is currently managed with multidisciplinary treatment strategies including surgery, radiation, chemotherapy, and targeted therapy. Drug resistance remains a continuing challenge, especially to targeted therapy utilizing monoclonal antibodies and tyrosine kinase inhibitors. This review discusses some of the recent molecular mechanisms that are involved in the development of resistance to Her2-targeted therapies including the PI3K/Akt/mTOR pathway, IGF-IR, Src, c-MET, the PP2A family, CD36, p27^kip1_, and miRNAs.

Keywords

HER2+ / Targeted therapy resistance / IGF-IR / Src / c-MET / PP2A / CD36 / miRNA

References

[1] Siegel RL,Fuchs HE.Cancer statistics, 2021.CA Cancer J Clin2021;71:7-33

[2] Kohler BA,Howlader N.Annual report to the nation on the status of cancer, 1975-2011, featuring incidence of breast cancer subtypes by race/ethnicity, poverty, and state.J Natl Cancer Inst2015;107:djv048 PMCID:PMC4603551

[3] WHO. Breast cancer. Available from: https://www.who.int/news-room/fact-sheets/detail/breast-cancer [Last accessed on 2 Apr 2022]

[4] Kalimutho M,Mittal D.Targeted therapies for triple-negative breast cancer: combating a stubborn disease.Trends Pharmacol Sci2015;36:822-46

[5] Yarden Y.Untangling the ErbB signalling network.Nat Rev Mol Cell Biol2001;2:127-37

[6] Lax I,Bellot F.Localization of a major receptor-binding domain for epidermal growth factor by affinity labeling.Mol Cell Biol1988;8:1831-4 PMCID:PMC363348

[7] Hsu JL.The role of HER2, EGFR, and other receptor tyrosine kinases in breast cancer.Cancer Metastasis Rev2016;35:575-88 PMCID:PMC5215954

[8] Olayioye MA.Update on HER-2 as a target for cancer therapy: intracellular signaling pathways of ErbB2/HER-2 and family members.Breast Cancer Res2001;3:385-9 PMCID:PMC138705

[9] Citri A.EGF-ERBB signalling: towards the systems level.Nat Rev Mol Cell Biol2006;7:505-16

[10] Moasser MM.The oncogene HER2: its signaling and transforming functions and its role in human cancer pathogenesis.Oncogene2007;26:6469-87 PMCID:PMC3021475

[11] Slamon DJ,Wong SG.Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene.Science1987;235:177-82

[12] Lyerly HK.The breast: comprehensive management of benign and malignant diseases.Ann Surg1992;215:192

[13] Dawood S,Buzdar AU,Giordano SH.Prognosis of women with metastatic breast cancer by HER2 status and trastuzumab treatment: an institutional-based review.J Clin Oncol2010;28:92-8 PMCID:PMC2799236

[14] Perez EA,Suman VJ.Trastuzumab plus adjuvant chemotherapy for human epidermal growth factor receptor 2-positive breast cancer: planned joint analysis of overall survival from NSABP B-31 and NCCTG N9831.JCO2014;32:3744-52 PMCID:PMC4226805

[15] Slamon DJ,Shak S.Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2.N Engl J Med2001;344:783-92

[16] Baselga J,Kim SB.CLEOPATRA Study GroupPertuzumab plus trastuzumab plus docetaxel for metastatic breast cancer.N Engl J Med2012;366:109-19 PMCID:PMC5705202

[17] Nielsen DL,Kamby C.HER2-targeted therapy in breast cancer. Monoclonal antibodies and tyrosine kinase inhibitors.Cancer Treat Rev2009;35:121-36

[18] Wong AL.Mechanisms of resistance to trastuzumab and novel therapeutic strategies in HER2-positive breast cancer.Int J Breast Cancer2012;2012:415170 PMCID:PMC3357513

[19] Hurvitz SA,O'Brien N.Current approaches and future directions in the treatment of HER2-positive breast cancer.Cancer Treat Rev2013;39:219-29 PMCID:PMC3835685

[20] Berns K,Hennessy BT.A functional genetic approach identifies the PI3K pathway as a major determinant of trastuzumab resistance in breast cancer.Cancer Cell2007;12:395-402

[21] Eichhorn PJ,Scaltriti M.Phosphatidylinositol 3-kinase hyperactivation results in lapatinib resistance that is reversed by the mTOR/phosphatidylinositol 3-kinase inhibitor NVP-BEZ235.Cancer Res2008;68:9221-30 PMCID:PMC2587064

[22] Margariti N,Bottini A.“Overcoming breast cancer drug resistance with mTOR inhibitors”. Could it be a myth or a real possibility in the short-term future?.Breast Cancer Res Treat2011;128:599-606

[23] Yuan R,Berg WJ.Targeting tumorigenesis: development and use of mTOR inhibitors in cancer therapy.J Hematol Oncol2009;2:45 PMCID:PMC2775749

[24] Martino MC, van Koetsveld PM, Pivonello R, Hofland LJ. Role of the mTOR pathway in normal and tumoral adrenal cells.Neuroendocrinology2010;92 Suppl 1:28-34

[25] Keck S,Rugo HS.Everolimus and its role in hormone-resistant and trastuzumab-resistant metastatic breast cancer.Future Oncol2012;8:1383-96

[26] Mayer I.Role of mTOR inhibition in preventing resistance and restoring sensitivity to hormone-targeted and HER2-targeted therapies in breast cancer.Clin Adv Hematol Oncol2013;11:217-24 PMCID:PMC3774138

[27] Genome Atlas Network. Comprehensive molecular portraits of human breast tumours.Nature2012;490:61-70 PMCID:PMC3465532

[28] Esteva FJ,Zhang S.PTEN, PIK3CA, p-AKT, and p-p70S6K status: association with trastuzumab response and survival in patients with HER2-positive metastatic breast cancer.Am J Pathol2010;177:1647-56 PMCID:PMC2947262

[29] Nagata Y,Zhou X.PTEN activation contributes to tumor inhibition by trastuzumab, and loss of PTEN predicts trastuzumab resistance in patients.Cancer Cell2004;6:117-27

[30] O'Brien NA,Chow L.Activated phosphoinositide 3-kinase/AKT signaling confers resistance to trastuzumab but not lapatinib.Mol Cancer Ther2010;9:1489-502

[31] Rexer BN,Dahlman K.Direct inhibition of PI3K in combination with dual HER2 inhibitors is required for optimal antitumor activity in HER2+ breast cancer cells.Breast Cancer Res2014;16:R9 PMCID:PMC3978602

[32] Baselga J,Im SA.Biomarker analyses in CLEOPATRA: a phase III, placebo-controlled study of pertuzumab in human epidermal growth factor receptor 2-positive, first-line metastatic breast cancer.J Clin Oncol2014;32:3753-61

[33] Baselga J,Verma S.Relationship between tumor biomarkers and efficacy in EMILIA, a phase III study of trastuzumab emtansine in HER2-positive metastatic breast cancer.Clin Cancer Res2016;22:3755-63 PMCID:PMC5485412

[34] Salgia R.MET in Lung cancer: biomarker selection based on scientific rationale.Mol Cancer Ther2017;16:555-65

[35] Fu YT,Zhou L.Valproic acid, targets papillary thyroid cancer through inhibition of c-Met signalling pathway. Am J Transl Res 2017;9:3138-47. PMCID:PMC5489911

[36] Bottaro DP,Faletto DL.Identification of the hepatocyte growth factor receptor as the c-met proto-oncogene product.Science1991;251:802-4

[37] Organ SL.An overview of the c-MET signaling pathway.Ther Adv Med Oncol2011;3:S7-S19 PMCID:PMC3225017

[38] Boccaccio C.Invasive growth: a MET-driven genetic programme for cancer and stem cells.Nat Rev Cancer2006;6:637-45

[39] Blumenschein GR Jr,Gonzalez-Angulo AM.Targeting the hepatocyte growth factor-cMET axis in cancer therapy.J Clin Oncol2012;30:3287-96 PMCID:PMC3434988

[40] Pilotto S,Karachaliou N.Tracking MET de-addiction in lung cancer: a road towards the oncogenic target.Cancer Treat Rev2017;60:1-11

[41] Zhang Y,Jin K.Function of the c-Met receptor tyrosine kinase in carcinogenesis and associated therapeutic opportunities.Mol Cancer2018;17:45 PMCID:PMC5817860

[42] Shattuck DL,Carraway KL 3rd.Met receptor contributes to trastuzumab resistance of Her2-overexpressing breast cancer cells.Cancer Res2008;68:1471-7

[43] Gastaldi S,Trusolino L.The Met oncogene and basal-like breast cancer: another culprit to watch out for?.Breast Cancer Res2010;12:208 PMCID:PMC2949647

[44] Beviglia L,Lin C,Kramer RH.Expression of the C-Met/HGF receptor in human breast carcinoma: correlation with tumor progression.Int J Cancer1997;74:301-9

[45] Raghav KP,Liu S.cMET and phospho-cMET protein levels in breast cancers and survival outcomes.Clin Cancer Res2012;18:2269-77 PMCID:PMC3821167

[46] Engelman JA,Mitsudomi T.MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling.Science2007;316:1039-43

[47] Pohlmann PR,Mernaugh R.Resistance to trastuzumab in breast cancer.Clin Cancer Res2009;15:7479-91 PMCID:PMC3471537

[48] Yue D.miR-182 regulates trastuzumab resistance by targeting Met in breast cancer cells.Cancer Gene Ther2019;26:1-10

[49] Liu L,Liu Y.Synergistic effects of foretinib with HER-targeted agents in MET and HER1- or HER2-coactivated tumor cells.Mol Cancer Ther2011;10:518-30

[50] Melo Gagliato D, Jardim DL, Marchesi MS, Hortobagyi GN. Mechanisms of resistance and sensitivity to anti-HER2 therapies in HER2+ breast cancer.Oncotarget2016;7:64431-46 PMCID:PMC5325455

[51] Lu Y,Zhao Y,Pollak M.Insulin-like growth factor-I receptor signaling and resistance to trastuzumab (Herceptin).J Natl Cancer Inst2001;93:1852-7

[52] Wang Y,Williams D.Inhibition of insulin-like growth factor-I receptor (IGF-IR) signaling and tumor cell growth by a fully human neutralizing anti-IGF-IR antibody.Mol Cancer Ther2005;4:1214-21

[53] Nahta R,Zhang B,Esteva FJ.Insulin-like growth factor-I receptor/human epidermal growth factor receptor 2 heterodimerization contributes to trastuzumab resistance of breast cancer cells.Cancer Res2005;65:11118-28

[54] Lu Y,Pollak M.Molecular mechanisms underlying IGF-I-induced attenuation of the growth-inhibitory activity of trastuzumab (Herceptin) on SKBR3 breast cancer cells.Int J Cancer2004;108:334-41

[55] Agnantis N,Zagorianakou P.Alterations of the cell cycle regulating proteins in invasive breast cancer.Molecular Genetics; Lung and Breast Carcinomas2002;1:425-38

[56] Nahta R,Ueno NT,Esteva FJ.P27(kip1) down-regulation is associated with trastuzumab resistance in breast cancer cells.Cancer Res2004;64:3981-6

[57] Hubalek M,Matthä K.Resistance to HER2-targeted therapy: mechanisms of trastuzumab resistance and possible strategies to overcome unresponsiveness to treatment.Wien Med Wochenschr2010;160:506-12

[58] Lloyd RV,Jin L.p27kip1: a multifunctional cyclin-dependent kinase inhibitor with prognostic significance in human cancers.The American Journal of Pathology1999;154:313-23 PMCID:PMC1850003

[59] Sherr CJ.CDK inhibitors: positive and negative regulators of G1-phase progression.Genes Dev1999;13:1501-12

[60] Guan X,Xie R.p27(Kip1) as a prognostic factor in breast cancer: a systematic review and meta-analysis.J Cell Mol Med2010;14:944-53 PMCID:PMC3823126

[61] Nakayama KI.Ubiquitin ligases: cell-cycle control and cancer.Nat Rev Cancer2006;6:369-81

[62] Lane HA,Beuvink I.Modulation of p27/Cdk2 complex formation through 4D5-mediated inhibition of HER2 receptor signaling.Ann Oncol2001;12 Suppl 1:S21-2

[63] Le XF,Lammayot A.The role of cyclin-dependent kinase inhibitor p27Kip1 in anti-HER2 antibody-induced G1 cell cycle arrest and tumor growth inhibition.J Biol Chem2003;278:23441-50

[64] Yakes FM,Ritter CA.Herceptin-induced inhibition of phosphatidylinositol-3 kinase and Akt Is required for antibody-mediated effects on p27, cyclin D1, and antitumor action. Cancer Res 2002;62:4132-41.

[65] Kute T,Willingham M.Development of herceptin resistance in breast cancer cells.Cytometry A2004;57:86-93

[66] Ishizawar R.c-Src and cooperating partners in human cancer.Cancer Cell2004;6:209-14

[67] Chu I,Arnaout A.p27 phosphorylation by Src regulates inhibition of cyclin E-Cdk2.Cell2007;128:281-94 PMCID:PMC1961623

[68] Bloom J.Deregulated degradation of the cdk inhibitor p27 and malignant transformation.Seminars in Cancer Biology2003;13:41-7

[69] Calin GA.MicroRNA signatures in human cancers.Nat Rev Cancer2006;6:857-66

[70] Galardi S,Giorda E.miR-221 and miR-222 expression affects the proliferation potential of human prostate carcinoma cell lines by targeting p27Kip1.J Biol Chem2007;282:23716-24

[71] Fornari F,Ferracin M.MiR-221 controls CDKN1C/p57 and CDKN1B/p27 expression in human hepatocellular carcinoma.Oncogene2008;27:5651-61

[72] Huynh TK,Chen JY.miR-221 confers lapatinib resistance by negatively regulating p27^kip1 in HER2-positive breast cancer.Cancer Sci2021;112:4234-45 PMCID:PMC8486195

[73] Yeatman TJ.A renaissance for SRC.Nat Rev Cancer2004;4:470-80

[74] Thomas SM.Cellular functions regulated by Src family kinases.Annu Rev Cell Dev Biol1997;13:513-609

[75] Kim LC,Haura EB.Src kinases as therapeutic targets for cancer.Nat Rev Clin Oncol2009;6:587-95

[76] Ralston R.The product of the protooncogene c-Src is modified during the cellular response to platelet-derived growth factor.Proc Natl Acad Sci U S A1985;82:7845-9 PMCID:PMC390866

[77] Weernink PA.Activation and translocation of c-Src to the cytoskeleton by both platelet-derived growth factor and epidermal growth factor.J Biol Chem1995;270:2264-7

[78] Zhang S,Li P.Combating trastuzumab resistance by targeting SRC, a common node downstream of multiple resistance pathways.Nat Med2011;17:461-9 PMCID:PMC3877934

[79] Peiró G,Gallardo A.Src, a potential target for overcoming trastuzumab resistance in HER2-positive breast carcinoma.Br J Cancer2014;111:689-95 PMCID:PMC4134494

[80] Ocana A,Antolín S.Efficacy and safety of dasatinib with trastuzumab and paclitaxel in first line HER2-positive metastatic breast cancer: results from the phase II GEICAM/2010-04 study.Breast Cancer Res Treat2019;174:693-701

[81] Formisano L,Rosa R.Epidermal growth factor-receptor activation modulates Src-dependent resistance to lapatinib in breast cancer models.Breast Cancer Res2014;16:R45 PMCID:PMC4076622

[82] Lu Y,Liu JH.Src family protein-tyrosine kinases alter the function of PTEN to regulate phosphatidylinositol 3-kinase/AKT cascades.J Biol Chem2003;278:40057-66

[83] O'Connor CM,Leonard D,Narla G.Therapeutic targeting of PP2A.Int J Biochem Cell Biol2018;96:182-93 PMCID:PMC5927617

[84] Pallas DC,Martin BL.Polyoma small and middle T antigens and SV40 small t antigen form stable complexes with protein phosphatase 2A.Cell1990;60:167-76

[85] Chen W,Campbell K.Identification of specific PP2A complexes involved in human cell transformation.Cancer Cell2004;5:127-36

[86] Wandzioch E,Werda A.PME-1 modulates protein phosphatase 2A activity to promote the malignant phenotype of endometrial cancer cells.Cancer Res2014;74:4295-305

[87] Sablina AA,Colpaert N.Identification of PP2A complexes and pathways involved in cell transformation.Cancer Res2010;70:10474-84 PMCID:PMC3056544

[88] Tan J,Li Z.B55β-associated PP2A complex controls PDK1-directed myc signaling and modulates rapamycin sensitivity in colorectal cancer.Cancer Cell2010;18:459-71

[89] Virshup DM.From promiscuity to precision: protein phosphatases get a makeover.Mol Cell2009;33:537-45

[90] Schmoldt A,Haberland G.Digitoxin metabolism by rat liver microsomes.Biochem Pharmacol1975;24:1639-41

[91] Haesen D,Derua R.Recurrent PPP2R1A mutations in uterine cancer act through a dominant-negative mechanism to promote malignant cell growth.Cancer Res2016;76:5719-31

[92] Curtis C,Chin SF.METABRIC GroupThe genomic and transcriptomic architecture of 2,000 breast tumours reveals novel subgroups.Nature2012;486:346-52 PMCID:PMC3440846

[93] Cheng AS,Chen Y.EZH2-mediated concordant repression of Wnt antagonists promotes β-catenin-dependent hepatocarcinogenesis.Cancer Res2011;71:4028-39

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

[95] Fingar DC,Tee AR.mTOR controls cell cycle progression through its cell growth effectors S6K1 and 4E-BP1/eukaryotic translation initiation factor 4E.Mol Cell Biol2004;24:200-16 PMCID:PMC303352

[96] Rodgers JT,Puigserver P.Clk2 and B56β mediate insulin-regulated assembly of the PP2A phosphatase holoenzyme complex on Akt.Mol Cell2011;41:471-9 PMCID:PMC3060660

[97] Hui L,Pielak RM.mTOR-dependent suppression of protein phosphatase 2A is critical for phospholipase D survival signals in human breast cancer cells.J Biol Chem2005;280:35829-35

[98] Peterson RT,Hardwick JS.Protein phosphatase 2A interacts with the 70-kDa S6 kinase and is activated by inhibition of FKBP12-rapamycinassociated protein.Proc Natl Acad Sci U S A1999;96:4438-42 PMCID:PMC16350

[99] Hahn K,Francis VA.PP2A regulatory subunit PP2A-B’ counteracts S6K phosphorylation.Cell Metab2010;11:438-44

[100] Padmanabhan S,Narasimhan SD.A PP2A regulatory subunit regulates C. elegans insulin/IGF-1 signaling by modulating AKT-1 phosphorylation.Cell2009;136:939-51 PMCID:PMC2707143

[101] Kuo YC,Yang CH.Regulation of phosphorylation of Thr-308 of Akt, cell proliferation, and survival by the B55alpha regulatory subunit targeting of the protein phosphatase 2A holoenzyme to Akt.J Biol Chem2008;283:1882-92

[102] Bao Y,Lee WC.EZH2-mediated PP2A inactivation confers resistance to HER2-targeted breast cancer therapy.Nat Commun2020;11:5878 PMCID:PMC7674491

[103] Tan JZ,Wang XX,Xu HE.EZH2: biology, disease, and structure-based drug discovery.Acta Pharmacol Sin2014;35:161-74 PMCID:PMC3914023

[104] Ookhtens M,Lyon I.Liver and adipose tissue contributions to newly formed fatty acids in an ascites tumor.Am J Physiol1984;247:R146-53

[105] Kamphorst JJ,Fan J.Hypoxic and Ras-transformed cells support growth by scavenging unsaturated fatty acids from lysophospholipids.Proc Natl Acad Sci U S A2013;110:8882-7 PMCID:PMC3670379

[106] Menendez JA.Fatty acid synthase and the lipogenic phenotype in cancer pathogenesis.Nat Rev Cancer2007;7:763-77

[107] Röhrig F.The multifaceted roles of fatty acid synthesis in cancer.Nat Rev Cancer2016;16:732-49

[108] Su X.Cellular fatty acid uptake: a pathway under construction.Trends Endocrinol Metab2009;20:72-7 PMCID:PMC2845711

[109] Febbraio M,Silverstein RL.CD36: a class B scavenger receptor involved in angiogenesis, atherosclerosis, inflammation, and lipid metabolism.J Clin Invest2001;108:785-91 PMCID:PMC200943

[110] Vazquez-Martin A,Brunet J.Pharmacological blockade of fatty acid synthase (FASN) reverses acquired autoresistance to trastuzumab (Herceptin by transcriptionally inhibiting ‘HER2 super-expression’ occurring in high-dose trastuzumab-conditioned SKBR3/Tzb100 breast cancer cells.Int J Oncol2007;31:769-76

[111] Feng WW,Kurokawa M.CD36: a key mediator of resistance to HER2 inhibitors in breast cancer.Mol Cell Oncol2020;7:1715766 PMCID:PMC7051136

[112] Silverstein RL.CD36, a scavenger receptor involved in immunity, metabolism, angiogenesis, and behavior.Sci Signal2009;2:re3 PMCID:PMC2811062

[113] Bartel DP.MicroRNAs: genomics, biogenesis, mechanism, and function.Cell2004;116:281-97

[114] Syeda Z, Langden SSS, Munkhzul C, Lee M, Song SJ. Regulatory mechanism of MicroRNA expression in cancer.Int J Mol Sci2020;21:1723 PMCID:PMC7084905

[115] Ahmad A,Kong D.Phosphoglucose isomerase/autocrine motility factor mediates epithelial-mesenchymal transition regulated by miR-200 in breast cancer cells.Cancer Res2011;71:3400-9 PMCID:PMC3085607

[116] Adams BD,White BA.Involvement of microRNAs in breast cancer.Semin Reprod Med2008;26:522-36

[117] Rao X,Li M.MicroRNA-221/222 confers breast cancer fulvestrant resistance by regulating multiple signaling pathways.Oncogene2011;30:1082-97 PMCID:PMC3342929

[118] Ye X,Zhu H.MiR-221 promotes trastuzumab-resistance and metastasis in HER2-positive breast cancers by targeting PTEN.BMB Rep2014;47:268-73 PMCID:PMC4163864

[119] Tian T,Feng X.MicroRNA-16 is putatively involved in the NF-κB pathway regulation in ulcerative colitis through adenosine A2a receptor (A2aAR) mRNA targeting.Sci Rep2016;6:30824 PMCID:PMC4967855

[120] Venturutti L,Rivas MA.MiR-16 mediates trastuzumab and lapatinib response in ErbB-2-positive breast and gastric cancer via its novel targets CCNJ and FUBP1.Oncogene2016;35:6189-202 PMCID:PMC5832962

[121] Corcoran C,Breslin S.miR-630 targets IGF1R to regulate response to HER-targeting drugs and overall cancer cell progression in HER2 over-expressing breast cancer.Mol Cancer2014;13:71 PMCID:PMC4234346

[122] Hunter FW,Lipert B.Mechanisms of resistance to trastuzumab emtansine (T-DM1) in HER2-positive breast cancer.Br J Cancer2020;122:603-12 PMCID:PMC7054312

[123] Nahta R,Hung MC,Esteva FJ.Mechanisms of disease: understanding resistance to HER2-targeted therapy in human breast cancer.Nat Clin Pract Oncol2006;3:269-80

[124] D'Amato V,Formisano L.Mechanisms of lapatinib resistance in HER2-driven breast cancer.Cancer Treat Rev2015;41:877-83

[125] Canfield K,Wilkins OM.Receptor tyrosine kinase ERBB4 mediates acquired resistance to ERBB2 inhibitors in breast cancer cells.Cell Cycle2015;14:648-55 PMCID:PMC4614407

[126] Garrett JT,Rinehart C.Transcriptional and posttranslational up-regulation of HER3 (ErbB3) compensates for inhibition of the HER2 tyrosine kinase.Proc Natl Acad Sci U S A2011;108:5021-6 PMCID:PMC3064360

[127] Sharial MSN, Crown J, Hennessy BT. Overcoming resistance and restoring sensitivity to HER2-targeted therapies in breast cancer.Ann Oncol2012;23:3007-16 PMCID:PMC3501233