Application of serum proteomics to the Women's Health Initiative conjugated equine estrogens trial reveals a multitude of effects relevant to clinical findings

Hiroyuki Katayama, Sophie Paczesny, Ross Prentice, Aaron Aragaki, Vitor M Faca, Sharon J Pitteri, Qing Zhang, Hong Wang, Melissa Silva, Jacob Kennedy, Jacques Rossouw, Rebecca Jackson, Judith Hsia, Rowan Chlebowski, Joann Manson, Samir Hanash, Hiroyuki Katayama, Sophie Paczesny, Ross Prentice, Aaron Aragaki, Vitor M Faca, Sharon J Pitteri, Qing Zhang, Hong Wang, Melissa Silva, Jacob Kennedy, Jacques Rossouw, Rebecca Jackson, Judith Hsia, Rowan Chlebowski, Joann Manson, Samir Hanash

Abstract

Background: The availability of serum collections from the Women's Health Initiative (WHI) conjugated equine estrogens (CEE) randomized controlled trial provides an opportunity to test the potential of in-depth quantitative proteomics to uncover changes in the serum proteome related to CEE and to assess their relevance to trial findings, including elevations in the risk of stroke and venous thromboembolism and a reduction in fractures.

Methods: Five independent large scale quantitative proteomics analyses were performed, each comparing a set of pooled serum samples collected from 10 subjects, 1 year following initiation of CEE at 0.625 mg/d, relative to their baseline pool. A subset of proteins that exhibited increased levels with CEE by quantitative proteomics was selected for validation studies.

Results: Of 611 proteins quantified based on differential stable isotope labeling, the levels of 116 (19%) were changed after 1 year of CEE (nominal P < 0.05), while 64 of these had estimated false discovery rates <0.05. Most of the changed proteins were not previously known to be affected by CEE and had relevance to processes that included coagulation, metabolism, osteogenesis, inflammation, and blood pressure maintenance. To validate quantitative proteomic data, 14 proteins were selected for ELISA. Findings for ten - IGF1, IGFBP4, IGFBP1, IGFBP2, F10, AHSG, GC, CP, MMP2, and PROZ - were confirmed in the initial set of 50 subjects and further validated in an independent set of 50 additional subjects who received CEE.

Conclusions: CEE affected a substantial fraction of the serum proteome, including proteins with relevance to findings from the WHI CEE trial related to cardiovascular disease and fracture.

Clinical trials registration: ClinicalTrials.gov identifier: NCT00000611.

Figures

Figure 1
Figure 1
Distribution of ratios for quantified peptides for the five IPAS experiments. A histogram of 1-year CEE/baseline (log2) ratios as determined from heavy-to-light isotopic labeling with acrylamide are shown for each IPAS experiment. The median of the distribution was centered at zero for normalization.
Figure 2
Figure 2
Volcano plots. (a) For nominal P-values. Relationship between the 1-year ET/baseline log2 ratios and their P-values. (b) For FDR adjusted P-values. Relationship between the 1 year ET/baseline log2 ratios and their FDR adjusted P-values.
Figure 3
Figure 3
Mean ratios (95% confidence intervals (CI)) for MS-based (IPAS, shown in red) and ELISA-based quantification (shown in black for the same set of 50 sera analyzed by MS and in blue for the independent set of 50 sera). SHBG ELISA data were based on a separate independent set of sera.
Figure 4
Figure 4
Comparison of mean ratios (1 year ET/baseline) by IPAS MS and by ELISA.
Figure 5
Figure 5
Dynamic range of IPAS MS pointing to proteins validated by ELISA. (a) Correlation between spectral counts (number of tandem mass spectra (MS2) acquired per protein) and estimated/measured serum concentrations. (b) Cumulative protein identifications are plotted versus ELISA protein concentration determined by ELISA measurments (red) and estimated concentration (blue) as determined by spectral counts.

References

    1. Marino M, Galluzzo P, Ascenzi P. Estrogen signaling multiple pathways to impact gene transcription. Curr Genomics. 2006;7:497–508. doi: 10.2174/138920206779315737.
    1. Lobo RA. Estrogen and cardiovascular disease. Ann N Y Acad Sci. 1990;592:286–294. doi: 10.1111/j.1749-6632.1990.tb30340.x. discussion 334-345.
    1. Stampfer MJ, Colditz GA. Estrogen replacement therapy and coronary heart disease: a quantitative assessment of the epidemiologic evidence. Prev Med. 1991;20:47–63. doi: 10.1016/0091-7435(91)90006-P.
    1. Anderson GL, Limacher M, Assaf AR, Bassford T, Beresford SA, Black H, Bonds D, Brunner R, Brzyski R, Caan B, Chlebowski R, Curb D, Gass M, Hays J, Heiss G, Hendrix S, Howard BV, Hsia J, Hubbell A, Jackson R, Johnson KC, Judd H, Kotchen JM, Kuller L, LaCroix AZ, Lane D, Langer RD, Lasser N, Lewis CE, Manson J. Effects of conjugated equine estrogen in postmenopausal women with hysterectomy: the Women's Health Initiative randomized controlled trial. JAMA. 2004;291:1701–1712. doi: 10.1001/jama.291.14.1701.
    1. Stefanick ML, Anderson GL, Margolis KL, Hendrix SL, Rodabough RJ, Paskett ED, Lane DS, Hubbell FA, Assaf AR, Sarto GE, Schenken RS, Yasmeen S, Lessin L, Chlebowski RT. Effects of conjugated equine estrogens on breast cancer and mammography screening in postmenopausal women with hysterectomy. JAMA. 2006;295:1647–1657. doi: 10.1001/jama.295.14.1647.
    1. Hsia J, Langer RD, Manson JE, Kuller L, Johnson KC, Hendrix SL, Pettinger M, Heckbert SR, Greep N, Crawford S, Eaton CB, Kostis JB, Caralis P, Prentice R. Conjugated equine estrogens and coronary heart disease: the Women's Health Initiative. Arch Intern Med. 2006;166:357–365. doi: 10.1001/archinte.166.3.357.
    1. Manson JE, Allison MA, Rossouw JE, Carr JJ, Langer RD, Hsia J, Kuller LH, Cochrane BB, Hunt JR, Ludlam SE, Pettinger MB, Gass M, Margolis KL, Nathan L, Ockene JK, Prentice RL, Robbins J, Stefanick ML. Estrogen therapy and coronary-artery calcification. N Engl J Med. 2007;356:2591–2602. doi: 10.1056/NEJMoa071513.
    1. Rossouw JE, Prentice RL, Manson JE, Wu L, Barad D, Barnabei VM, Ko M, LaCroix AZ, Margolis KL, Stefanick ML. Postmenopausal hormone therapy and risk of cardiovascular disease by age and years since menopause. JAMA. 2007;297:1465–1477. doi: 10.1001/jama.297.13.1465.
    1. Brunner RL, Gass M, Aragaki A, Hays J, Granek I, Woods N, Mason E, Brzyski R, Ockene J, Assaf A, LaCroix A, Matthews K, Wallace R. Effects of conjugated equine estrogen on health-related quality of life in postmenopausal women with hysterectomy: results from the Women's Health Initiative Randomized Clinical Trial. Arch Intern Med. 2005;165:1976–1986. doi: 10.1001/archinte.165.17.1976.
    1. Prentice RL, Anderson GL. The women's health initiative: lessons learned. Annu Rev Public Health. 2008;29:131–150. doi: 10.1146/annurev.publhealth.29.020907.090947.
    1. Nelson HD, Humphrey LL, Nygren P, Teutsch SM, Allan JD. Postmenopausal hormone replacement therapy: scientific review. JAMA. 2002;288:872–881. doi: 10.1001/jama.288.7.872.
    1. Curb JD, Prentice RL, Bray PF, Langer RD, Van Horn L, Barnabei VM, Bloch MJ, Cyr MG, Gass M, Lepine L, Rodabough RJ, Sidney S, Uwaifo GI, Rosendaal FR. Venous thrombosis and conjugated equine estrogen in women without a uterus. Arch Intern Med. 2006;166:772–780. doi: 10.1001/archinte.166.7.772.
    1. Hendrix SL, Wassertheil-Smoller S, Johnson KC, Howard BV, Kooperberg C, Rossouw JE, Trevisan M, Aragaki A, Baird AE, Bray PF, Buring JE, Criqui MH, Herrington D, Lynch JK, Rapp SR, Torner J. Effects of conjugated equine estrogen on stroke in the Women's Health Initiative. Circulation. 2006;113:2425–2434. doi: 10.1161/CIRCULATIONAHA.105.594077.
    1. Langer RD, Pradhan AD, Lewis CE, Manson JE, Rossouw JE, Hendrix SL, LaCroix AZ, Ridker PM. Baseline associations between postmenopausal hormone therapy and inflammatory, haemostatic, and lipid biomarkers of coronary heart disease. The Women's Health Initiative Observational Study. Thromb Haemost. 2005;93:1108–1116.
    1. Ichikawa J, Sumino H, Ichikawa S, Ozaki M. Different effects of transdermal and oral hormone replacement therapy on the renin-angiotensin system, plasma bradykinin level, and blood pressure of normotensive postmenopausal women. Am J Hypertens. 2006;19:744–749. doi: 10.1016/j.amjhyper.2005.10.006.
    1. Schunkert H, Danser AH, Hense HW, Derkx FH, Kurzinger S, Riegger GA. Effects of estrogen replacement therapy on the renin-angiotensin system in postmenopausal women. Circulation. 1997;95:39–45.
    1. Andersson I, Aspergren K, Janzon L, Landberg T, Lindholm K, Linell F, Ljungberg O, Ranstam J, Sigfusson B. Mammographic screening and mortality from breast cancer: the Malmo mammographic screening trial. BMJ. 1988;297:943–948. doi: 10.1136/bmj.297.6654.943.
    1. Heald A, Kaushal K, Anderson S, Redpath M, Durrington PN, Selby PL, Gibson MJ. Effects of hormone replacement therapy on insulin-like growth factor (IGF)-I, IGF-II and IGF binding protein (IGFBP)-1 to IGFBP-4: implications for cardiovascular risk. Gynecol Endocrinol. 2005;20:176–182. doi: 10.1080/09513590400027406.
    1. Faca V, Coram M, Phanstiel D, Glukhova V, Zhang Q, Fitzgibbon M, McIntosh M, Hanash S. Quantitative analysis of acrylamide labeled serum proteins by LC-MS/MS. J Proteome Res. 2006;5:2009–2018. doi: 10.1021/pr060102+.
    1. Faca V, Pitteri SJ, Newcomb L, Glukhova V, Phanstiel D, Krasnoselsky A, Zhang Q, Struthers J, Wang H, Eng J, Fitzgibbon M, McIntosh M, Hanash S. Contribution of protein fractionation to depth of analysis of the serum and plasma proteomes. J Proteome Res. 2007;6:3558–3565. doi: 10.1021/pr070233q.
    1. Hanash SM, Pitteri SJ, Faca VM. Mining the plasma proteome for cancer biomarkers. Nature. 2008;452:571–579. doi: 10.1038/nature06916.
    1. Faca VM, Song KS, Wang H, Zhang Q, Krasnoselsky AL, Newcomb LF, Plentz RR, Gurumurthy S, Redston MS, Pitteri SJ, Pereira-Faca SR, Ireton RC, Katayama H, Glukhova V, Phanstiel D, Brenner DE, Anderson MA, Misek D, Scholler N, Urban ND, Barnett MJ, Edelstein C, Goodman GE, Thornquist MD, McIntosh MW, DePinho RA, Bardeesy N, Hanash SM. A mouse to human search for plasma proteome changes associated with pancreatic tumor development. PLoS Med. 2008;5:e123. doi: 10.1371/journal.pmed.0050123.
    1. Gericke B, Koebnick C, Reimann M, Forterre S, Franz Zunft HJ, Schweigert FJ. Influence of hormone replacement therapy on proteomic pattern in serum of postmenopausal women. Maturitas. 2005;51:334–342. doi: 10.1016/j.maturitas.2004.08.016.
    1. Pitteri SJ, Faca VM, Kelly-Spratt KS, Kasarda AE, Wang H, Zhang Q, Newcomb L, Krasnoselesky A, Paczesny S, Choi G, Fitzgibbon M, McIntosh MW, Kemp CJ, Hanash SM. Plasma proteome profiling of a mouse model of breast cancer identifies a set of up-regulated proteins in common with human breast cancer cells. J Proteome Res. 2008;7:1481–1489. doi: 10.1021/pr7007994.
    1. Rauch A, Bellew M, Eng J, Fitzgibbon M, Holzman T, Hussey P, Igra M, Maclean B, Lin CW, Detter A, Fang R, Faca V, Gafken P, Zhang H, Whiteaker J, States D, Hanash S, Paulovich A, McIntosh MW. Computational Proteomics Analysis System (CPAS): an extensible, open-source analytic system for evaluating and publishing proteomic data and high throughput biological experiments. J Proteome Res. 2006;5:112–121. doi: 10.1021/pr0503533.
    1. Keller A, Nesvizhskii AI, Kolker E, Aebersold R. Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search. Anal Chem. 2002;74:5383–5392. doi: 10.1021/ac025747h.
    1. Nesvizhskii AI, Keller A, Kolker E, Aebersold R. A statistical model for identifying proteins by tandem mass spectrometry. Anal Chem. 2003;75:4646–4658. doi: 10.1021/ac0341261.
    1. R Development Core Team. R A language and environment for statistical computing. R Foundation for Statistical Computing. Vienna, Austria; 2007.
    1. Smyth GK. Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat Appl Genet Mol Biol. 2004;3:Article3.
    1. Smyth GK. In: Bioinformatics and Computational Biology Solutions using R and Bioconductor. Gentleman R, Carey V, Dudoit S, Irizarry R, Huber W, editor. New York: Springer; 2005. Limma: linear models for microarray data. pp. 397–420. full_text.
    1. Benjamini Y, Hochberg Y. Controlling false discovery rate: a practical and powerful approach to multiple testing. J Roy Stat Soc. 1995;57:289–300.
    1. Nikolsky Y, Ekins S, Nikolskaya T, Bugrim A. A novel method for generation of signature networks as biomarkers from complex high throughput data. Toxicol Lett. 2005;158:20–29. doi: 10.1016/j.toxlet.2005.02.004.
    1. Jarvinen AK, Autio R, Haapa-Paananen S, Wolf M, Saarela M, Grenman R, Leivo I, Kallioniemi O, Makitie AA, Monni O. Identification of target genes in laryngeal squamous cell carcinoma by high-resolution copy number and gene expression microarray analyses. Oncogene. 2006;25:6997–7008. doi: 10.1038/sj.onc.1209690.
    1. Lee TL, Alba D, Baxendale V, Rennert OM, Chan WY. Application of transcriptional and biological network analyses in mouse germ-cell transcriptomes. Genomics. 2006;88:18–33. doi: 10.1016/j.ygeno.2006.03.008.
    1. Vogelstein B, Kinzler KW. Cancer genes and the pathways they control. Nat Med. 2004;10:789–799. doi: 10.1038/nm1087.
    1. Bourdeau V, Deschenes J, Metivier R, Nagai Y, Nguyen D, Bretschneider N, Gannon F, White JH, Mader S. Genome-wide identification of high-affinity estrogen response elements in human and mouse. Mol Endocrinol. 2004;18:1411–1427. doi: 10.1210/me.2003-0441.
    1. Pentecost BT, Teng CT. Lactotransferrin is the major estrogen inducible protein of mouse uterine secretions. J Biol Chem. 1987;262:10134–10139.
    1. Teng CT, Walker MP, Bhattacharyya SN, Klapper DG, DiAugustine RP, McLachlan JA. Purification and properties of an oestrogen-stimulated mouse uterine glycoprotein (approx. 70 kDa). Biochem J. 1986;240:413–422.
    1. Teng CT. Lactoferrin gene expression and regulation: an overview. Biochem Cell Biol. 2002;80:7–16. doi: 10.1139/o01-215.
    1. Teng CT. Factors regulating lactoferrin gene expression. Biochem Cell Biol. 2006;84:263–267. doi: 10.1139/O06-034.
    1. Zhao YY, Zhou J, Narayanan CS, Cui Y, Kumar A. Role of C/A polymorphism at -20 on the expression of human angiotensinogen gene. Hypertension. 1999;33:108–115.
    1. May FE, Westley BR. Expression of human intestinal trefoil factor in malignant cells and its regulation by oestrogen in breast cancer cells. J Pathol. 1997;182:404–413. doi: 10.1002/(SICI)1096-9896(199708)182:4<404::AID-PATH875>;2-0.
    1. GeneCards: TFF3 Gene.
    1. Gronbaek H, Vestergaard EM, Hey H, Nielsen JN, Nexo E. Serum trefoil factors in patients with inflammatory bowel disease. Digestion. 2006;74:33–39. doi: 10.1159/000096591.
    1. Vestergaard EM, Poulsen SS, Gronbaek H, Larsen R, Nielsen AM, Ejskjaer K, Clausen JT, Thim L, Nexo E. Development and evaluation of an ELISA for human trefoil factor 3. Clin Chem. 2002;48:1689–1695.
    1. Westley B, Rochefort H. A secreted glycoprotein induced by estrogen in human breast cancer cell lines. Cell. 1980;20:353–362. doi: 10.1016/0092-8674(80)90621-2.
    1. Puistola U, Westerlund A, Kauppila A, Turpeenniemi-Hujanen T. Regulation of 72-kd type IV collagenase-matrix metalloproteinase-2 by estradiol and gonadotropin-releasing hormone agonist in human granulosa-lutein cells. Fertil Steril. 1995;64:81–87.
    1. Marin-Castano ME, Elliot SJ, Potier M, Karl M, Striker LJ, Striker GE, Csaky KG, Cousins SW. Regulation of estrogen receptors and MMP2 expression by estrogens in human retinal pigment epithelium. Invest Ophthalmol Vis Sci. 2003;44:50–59. doi: 10.1167/iovs.01-1276.
    1. Qin H, Sun Y, Benveniste EN. The transcription factors Sp1, Sp3, and AP-2 are required for constitutive matrix metalloproteinase-2 gene expression in astroglioma cells. J Biol Chem. 1999;274:29130–29137. doi: 10.1074/jbc.274.41.29130.
    1. Bian J, Sun Y. Transcriptional activation by p53 of the human type IV collagenase (gelatinase A or matrix metalloproteinase 2) promoter. Mol Cell Biol. 1997;17:6330–6338.
    1. Kooperberg C, Cushman M, Hsia J, Robinson JG, Aragaki AK, Lynch JK, Baird AE, Johnson KC, Kuller LH, Beresford SA, Rodriguez B. Can biomarkers identify women at increased stroke risk? The Women's Health Initiative Hormone Trials. PLoS Clin Trials. 2007;2:e28. doi: 10.1371/journal.pctr.0020028.
    1. Fossum S, Hoem NO, Gjonnaess H, Briseid K. Contact activation factors in plasma from women on estrogen replacement therapy after ovariohysterectomy. Thromb Res. 1999;93:161–170. doi: 10.1016/S0049-3848(98)00183-2.
    1. McQuillan AM, Eikelboom JW, Hankey GJ, Baker R, Thom J, Staton J, Yi Q, Cole V. Protein Z in ischemic stroke and its etiologic subtypes. Stroke. 2003;34:2415–2419. doi: 10.1161/01.STR.0000092124.52084.4B.
    1. Galis ZS, Kranzhofer R, Fenton JW 2nd, Libby P. Thrombin promotes activation of matrix metalloproteinase-2 produced by cultured vascular smooth muscle cells. Arterioscler Thromb Vasc Biol. 1997;17:483–489.
    1. Humphries SE, Whittall RA, Hubbart CS, Maplebeck S, Cooper JA, Soutar AK, Naoumova R, Thompson GR, Seed M, Durrington PN, Miller JP, Betteridge DJ, Neil HA. Genetic causes of familial hypercholesterolaemia in patients in the UK: relation to plasma lipid levels and coronary heart disease risk. J Med Genet. 2006;43:943–949. doi: 10.1136/jmg.2006.038356.
    1. Abifadel M, Varret M, Rabes JP, Allard D, Ouguerram K, Devillers M, Cruaud C, Benjannet S, Wickham L, Erlich D, Derre A, Villeger L, Farnier M, Beucler I, Bruckert E, Chambaz J, Chanu B, Lecerf JM, Luc G, Moulin P, Weissenbach J, Prat A, Krempf M, Junien C, Seidah NG, Boileau C. Mutations in PCSK9 cause autosomal dominant hypercholesterolemia. Nat Genet. 2003;34:154–156. doi: 10.1038/ng1161.
    1. Wallentin L, Larsson-Cohn U. Metabolic and hormonal effects of post-menopausal oestrogen replacement treatment. II. Plasma lipids. Acta Endocrinol. 1977;86:597–607.
    1. Fernandez-Real JM, Pugeat M, Grasa M, Broch M, Vendrell J, Brun J, Ricart W. Serum corticosteroid-binding globulin concentration and insulin resistance syndrome: a population study. J Clin Endocrinol Metab. 2002;87:4686–4690. doi: 10.1210/jc.2001-011843.
    1. Hashimoto S, Miwa M, Akasofu K, Nishida E. Changes in 40 serum proteins of post-menopausal women. Maturitas. 1991;13:23–33. doi: 10.1016/0378-5122(91)90282-U.
    1. Iyengar S, Hamman RF, Marshall JA, Majumder PP, Ferrell RE. On the role of vitamin D binding globulin in glucose homeostasis: results from the San Luis Valley Diabetes Study. Genet Epidemiol. 1989;6:691–698. doi: 10.1002/gepi.1370060606.
    1. Ikeda Y, Imai Y, Kumagai H, Nosaka T, Morikawa Y, Hisaoka T, Manabe I, Maemura K, Nakaoka T, Imamura T, Miyazono K, Komuro I, Nagai R, Kitamura T. Vasorin, a transforming growth factor beta-binding protein expressed in vascular smooth muscle cells, modulates the arterial response to injury in vivo. Proc Natl Acad Sci USA. 2004;101:10732–10737. doi: 10.1073/pnas.0404117101.
    1. Govers-Riemslag JW, Smid M, Cooper JA, Bauer KA, Rosenberg RD, Hack CE, Hamulyak K, Spronk HM, Miller GJ, ten Cate H. The plasma kallikrein-kinin system and risk of cardiovascular disease in men. J Thromb Haemost. 2007;5:1896–1903. doi: 10.1111/j.1538-7836.2007.02687.x.
    1. Sonnet E, Lacut K, Roudaut N, Mottier D, Kerlan V, Oger E. Effects of the route of oestrogen administration on IGF-1 and IGFBP-3 in healthy postmenopausal women: results from a randomized placebo-controlled study. Clin Endocrinol (Oxf) 2007;66:626–631. doi: 10.1111/j.1365-2265.2007.02783.x.
    1. Taskinen MR, Puolakka J, Pyorala T, Luotola H, Bjaorn M, Kaarianen J, Lahdenpera S, Ehnholm C. Hormone replacement therapy lowers plasma Lp(a) concentrations. Comparison of cyclic transdermal and continuous estrogen-progestin regimens. Arterioscler Thromb Vasc Biol. 1996;16:1215–1221.
    1. SymAtlas.
    1. Canonico M, Plu-Bureau G, Lowe GD, Scarabin PY. Hormone replacement therapy and risk of venous thromboembolism in postmenopausal women: systematic review and meta-analysis. Bmj. 2008;336:1227–1231. doi: 10.1136/.
    1. WHI Participant Website.
    1. Helgason S, Damber JE, Damber MG, von Schoultz B, Selstam G, Sodergard R. A comparative longitudinal study on sex hormone binding globulin capacity during estrogen replacement therapy. Acta Obstet Gynecol Scand. 1982;61:97–100. doi: 10.3109/00016348209156536.
    1. Kelver ME, Kaul A, Nowicki B, Findley WE, Hutchens TW, Nagamani M. Estrogen regulation of lactoferrin expression in human endometrium. Am J Reprod Immunol. 1996;36:243–247.

Source: PubMed

Szponzorok és közreműködők

Egészségi állapot

Kábítószer-beavatkozások

3
Iratkozz fel