Skip to main content

Advertisement

SpringerLink
Log in

Pilot and feasibility study: prospective proteomic profiling of mammary epithelial cells from high-risk women provides evidence of activation of pro-survival pathways

  • Preclinical study
  • Published:
Breast Cancer Research and Treatment Aims and scope Submit manuscript

An Erratum to this article was published on 31 August 2012

Abstract

Normal mammary gland homeostasis requires the coordinated regulation of protein signaling networks. However, we have little prospective information on whether activation of protein signaling occurs in premalignant mammary epithelial cells, as represented by cells with cytological atypia from women who are at high risk for breast cancer. This information is critical for understanding the role of deregulated signaling pathways in the initiation of breast cancer and for developing targeted prevention and/or treatment strategies for breast cancer in the future. In this pilot and feasibility study, we examined the expression of 52 phosphorylated, total, and cleaved proteins in 31 microdissected Random Periareolar Fine Needle Aspiration (RPFNA) samples by high-throughput Reverse Phase Protein Microarray. Unsupervised hierarchical clustering analysis indicated the presence of four clusters of proteins that represent the following signaling pathways: (1) receptor tyrosine kinase/Akt/mammalian target of rapamycin (RTK/Akt/mTOR), (2) RTK/Akt/extracellular signal-regulated kinase (RTK/Akt/ERK), (3) mitochondrial apoptosis, and (4) indeterminate. Clusters 1 through 3 comprised moderately to highly expressed proteins, while Cluster 4 comprised proteins that are lowly expressed in a majority of RPFNA samples. Our exploratory study showed that the interlinked components of mitochondrial apoptosis pathway are highly expressed in all mammary epithelial cells obtained from high-risk women. In particular, the expression levels of anti-apoptotic Bcl-xL and pro-apoptotic Bad are positively correlated in both non-atypical and atypical samples (unadjusted P < 0.0001), suggesting a delicate balance between the pro-apoptotic and anti-apoptotic regulation of cell proliferation during the early steps of mammary carcinogenesis. Our feasibility study suggests that the activation of key proteins along the RTK/Akt pathway may tip this balance to cell survival. Taken together, our results demonstrate the feasibility of mapping proteomic signaling networks in limited RPFNA samples obtained from high-risk women and the promise of developing rational drug targets or preventative strategies for breast cancer in future proteomic studies with a larger cohort of high-risk women.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Notes

  1. Original pathway map was obtained from SABiosciences Pathway Central (www.sabiosciences.com).

Abbreviations

Apaf-1:

Apoptotic protease activating factor-1

Bad:

Bcl2-antagonist of cell death

Bax:

Bcl2 associated X-protein

Bcl-2:

B-cell lymphoma-2

Bid:

Bcl2 interacting protein

BRCA1/2:

Breast cancer-associated gene 1/2

CK2:

Casein kinase II or 2

DCIS:

Ductal carcinoma in situ

EGFR:

Epidermal growth factor receptor

ER:

Estrogen receptor

ErbB:

Erythroblastic leukemia viral oncogene homolog

ERK:

Extracellular signal-regulated kinase

FADD:

Fas-associated via death domain

IHC:

Immunohistochemistry

IGF-1R:

Insulin-like growth factor receptor

LCIS:

Lobular carcinoma in situ

LCM:

Laser capture microdissection

mTOR:

Mammalian target of rapamycin

OR:

Operating room

PI3K:

Phosphatidylinositol 3-kinase

PTEN:

Phosphatase and tensin homolog

RPFNA:

Random Periareolar Fine Needle Aspiration

RPPM:

Reverse phase protein microarray

RTK:

Receptor tyrosine kinase

References

  1. Wickenden JA, Watson CJ (2010) Signalling downstream of PI3 kinase in mammary epithelium: a play in 3 Akts. Breast Cancer Res 12:202

    Article  PubMed  Google Scholar 

  2. Hynes NE, Watson CJ (2010) Mammary gland growth factors: roles in normal development and in cancer. Cold Spring Harb Perspect 2:a003186

    Article  Google Scholar 

  3. Brandt R, Eisenbrandt R, Leenders F, Zschiesche W, Binas B, Juergensen C, Theuring F (2000) Mammary gland specific hEGF receptor transgene expression induces neoplasia and inhibits differentiation. Oncogene 19:2129–2137

    Article  PubMed  CAS  Google Scholar 

  4. Wood TL, Richert MM, Stull MA, Allar MA (2000) The insulin-like growth factors (IGFs) and IGF binding proteins in postnatal development of murine mammary glands. J Mammary Gland Biol Neoplasia 5:31–42

    Article  PubMed  CAS  Google Scholar 

  5. Arboleda MJ, Lyons JF, Kabbinavar FF, Bray MR, Snow BE, Ayala R, Danino M, Karlan BY, Slamon DJ (2003) Overexpression of Akt2/protein kinase Bβ leads to up-regulation of β1 integrins, increased invasion, and metastasis of human breast and ovarian cancer cells. Cancer Res 63:196–206

    PubMed  CAS  Google Scholar 

  6. Hutchinson JN, Jin J, Cardiff RD, Woodgett JR, Muller WJ (2004) Activation of Akt-1 (PKB-alpha) can accelerate ErbB-2-mediated mammary tumorigenesis but suppresses tumor invasion. Cancer Res 64:3171–3178

    Article  PubMed  CAS  Google Scholar 

  7. Nakatani K, Thompson DA, Barthel A, Sakaue H, Liu W, Weigel RJ, Roth RA (1999) Up-regulation of Akt3 in estrogen receptor-deficient breast cancers and androgen-independent prostate cancer lines. J Biol Chem 274:21528–21532

    Article  PubMed  CAS  Google Scholar 

  8. Gulmann C, Sheehan KM, Kay EW, Liotta LA, Petricoin EF III (2006) Array-based proteomics: mapping of protein circuitries for diagnostics, prognostics, and therapy guidance in cancer. J Pathol 208:595–606

    Article  PubMed  CAS  Google Scholar 

  9. Petricoin EF III, Bichsel VE, Calvert VS, Espina V, Winters M, Young L, Belluco C, Trock BJ, Lippman M, Fishman DA, Sgroi DC, Munson PJ, Esserman LJ, Liotta LA (2005) Mapping molecular networks using proteomics: a vision for patient-tailored combination therapy. J Clin Oncol 23:3614–3621

    Article  PubMed  CAS  Google Scholar 

  10. Hait WN (2009) Targeted cancer therapeutics. Cancer Res 69:1263–1267

    Article  PubMed  CAS  Google Scholar 

  11. Espina V, Liotta LA, Petricoin EF III (2009) Reverse-phase protein microarrays for theranostics and patient tailored therapy. Methods Mol Biol 520:89–105

    Article  PubMed  CAS  Google Scholar 

  12. Paweletz CP, Charboneau L, Bichsel VE, Simone NL, Chen T, Gillespie JW, Emmert-Buck MR, Roth MJ, Petricoin EF III, Liotta LA (2001) Reverse phase protein microarrays which capture disease progression show activation of pro-survival pathways at the cancer invasion front. Oncogene 20:1981–1989

    Article  PubMed  CAS  Google Scholar 

  13. Petricoin EF III, Espina V, Araujo RP, Midure B, Yeung C, Wan X, Eichler GS, Johann DJ Jr, Qualman S, Tsokos M, Krishnan K, Helman LJ, Liotta LA (2007) Phosphoprotein pathway mapping: Akt/mammalian target of rapamycin activation is negatively associated with childhood rhabdomyosarcoma survival. Cancer Res 67:3431–3440

    Article  PubMed  CAS  Google Scholar 

  14. Fabian CJ, Kimler BF, Zalles CM, Klemp JR, Kamel S, Zeiger S, Mayo MS (2000) Short-term breast cancer prediction by random periareolar fine-needle aspiration cytology and the Gail risk model. J Natl Cancer Inst 92:1217–1227

    Article  PubMed  CAS  Google Scholar 

  15. Fabian CJ, Kimler BF, Brady DA, Mayo MS, Chang CH, Ferraro JA, Zalles CM, Stanton AL, Masood S, Grizzle WE, Boyd NF, Arneson DW, Johnson KA (2002) A phase II breast cancer chemoprevention trial of oral alpha-difluoromethylornithine: breast tissue, imaging, and serum and urine biomarkers. Clin Cancer Res 8:3105–3117

    PubMed  CAS  Google Scholar 

  16. Ibarra-Drendall C, Wilke LG, Zalles C, Scott V, Archer LE, Lem S, Yee LD, Lester J, Kulkarni S, Murekeyisoni C, Wood M, Wilson K, Garber J, Gentry C, Stouder A, Broadwater G, Baker JC Jr, Vasilatos SN, Owens E, Rabiner S, Barron AC, Seewaldt VL (2009) Reproducibility of random periareolar fine needle aspiration in a multi-institutional Cancer and Leukemia Group B (CALGB) cross-sectional study. Cancer Epidemiol Biomarkers Prev 18:1379–1385

    Article  PubMed  Google Scholar 

  17. Bean GR, Scott V, Yee L, Ratliff-Daniel B, Troch MM, Seo P, Bowie ML, Marcom PK, Slade J, Kimler BF, Fabian CJ, Zalles CM, Broadwater G, Baker JC Jr, Wilke LG, Seewaldt VL (2005) Retinoic acid receptor-beta2 promoter methylation in random periareolar fine needle aspiration. Cancer Epidemiol Biomarkers Prev 14:790–798

    Article  PubMed  CAS  Google Scholar 

  18. Bean GR, Ibarra Drendall C, Goldenberg VK, Baker JC Jr, Troch MM, Paisie C, Wilke LG, Yee L, Marcom PK, Kimler BF, Fabian CJ, Zalles CM, Broadwater G, Scott V, Seewaldt VL (2007) Hypermethylation of the breast cancer-associated gene 1 promoter does not predict cytologic atypia or correlate with surrogate end points of breast cancer risk. Cancer Epidemiol Biomarkers Prev 16:50–56

    Article  PubMed  CAS  Google Scholar 

  19. Zalles C, Kimler BF, Kamel S, McKittrick R, Fabian CJ (1995) Cytology patterns in random aspirates from women at high and low risk for breast cancer. Breast J 1:343–349

    Article  Google Scholar 

  20. Masood S, Frykberg ER, McLellan GL, Scalapino MC, Mitchum DG, Bullard JB (1990) Prospective evaluation of radiologically directed fine-needle aspiration biopsy of nonpalpable breast lesions. Cancer 66:1480–1487

    Article  PubMed  CAS  Google Scholar 

  21. Barry WT, Nobel AB, Wright FA (2008) A statistical framework for testing functional categories in microarray data. Ann Appl Stat 2:286–315

    Article  Google Scholar 

  22. Skolnick MH, Cannon-Albright LA, Goldgar DE, Ward JH, Marshall CJ, Schumann GB, Hogle H, McWhorter WP, Wright EC, Tran TD et al (1990) Inheritance of proliferative breast disease in breast cancer kindreds. Science 250:1715–1720

    Article  PubMed  CAS  Google Scholar 

  23. Wrensch MR, Petrakis NL, Miike R, King EB, Chew K, Neuhaus J, Lee MM, Rhys M (2001) Breast cancer risk in women with abnormal cytology in nipple aspirates of breast fluid. J Natl Cancer Inst 93:1791–1798

    Article  PubMed  CAS  Google Scholar 

  24. Page DL, Dupont WD (1990) Anatomic markers of human premalignancy and risk of breast cancer. Cancer 66:1326–1335

    Article  PubMed  CAS  Google Scholar 

  25. Marshall LM, Hunter DJ, Connolly JL, Schnitt SJ, Byrne C, London SJ, Colditz GA (1997) Risk of breast cancer associated with atypical hyperplasia of lobular and ductual types. Cancer Epidemiol Biomarkers Prev 6:297–301

    PubMed  CAS  Google Scholar 

  26. Hengartner MO (2000) The biochemistry of apoptosis. Nature 407:770–776

    Article  PubMed  CAS  Google Scholar 

  27. Youle RJ, Strasser A (2008) The BCL-2 protein family: opposing activities that mediate cell death. Nat Rev Mol Cell Biol 9:47–59

    Article  PubMed  CAS  Google Scholar 

  28. Hanahan D, Weinberg RA (2000) The hallmarks of cancer. Cell 100:57–70

    Article  PubMed  CAS  Google Scholar 

  29. Harris SL, Levine AJ (2005) The p53 pathway: positive and negative feedback loops. Oncogene 24:2899–2908

    Article  PubMed  CAS  Google Scholar 

  30. Pilie PG, Ibarra-Drendall C, Troch MM, Broadwater G, Barry WT, Petricoin EF III, Wulfkuhle JD, Liotta LA, Lem S, Baker JC Jr, Stouder A, Ford AC, Wilke LG, Zalles CM, Mehta P, Williams J, Shivraj M, Su Z, Geradts J, Yu D, Seewaldt VL (2011) Protein microarray analysis of mammary epithelial cells from obese and nonobese women at high risk for breast cancer: feasibility data. Cancer Epidemiol Biomarkers Prev 20:476–482

    Article  PubMed  CAS  Google Scholar 

  31. Foley J, Nickerson NK, Nam S et al (2010) EGFR signaling in breast cancer: bad to the bone. Semin Cell Dev Biol 21:951–960

    Article  PubMed  CAS  Google Scholar 

  32. Khwaja A, Rodriguez-Viciana P, Wennstrom S, Warne PH, Downward J (1997) Matrix adhesion and Ras transformation both activate a phosphoinositide 3-OH kinase and protein kinase B/Akt cellular survival pathway. EMBO J 16:2783–2793

    Article  PubMed  CAS  Google Scholar 

  33. Datta SR, Dudek H, Tao X, Masters S, Fu H, Gotoh Y, Greenberg ME (1997) Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell 91:231–241

    Article  PubMed  CAS  Google Scholar 

  34. Zha J, Harada H, Yang E, Jockel J, Korsmeyer SJ (1996) Serine phosphorylation of death agonist BAD in response to survival factor results in binding to 14-3-3 not BCL-X(L). Cell 87:619–628

    Article  PubMed  CAS  Google Scholar 

  35. Zha J, Harada H, Osipov K, Jockel J, Waksman G, Korsmeyer SJ (1997) BH3 domain of BAD is required for heterodimerization with BCL-XL and pro-apoptotic activity. J Biol Chem 272:24101–24104

    Article  PubMed  CAS  Google Scholar 

  36. Torres J, Pulido R (2001) The tumor suppressor PTEN is phosphorylated by protein kinase CK2 at its C terminus. Implications for PTEN stability to proteasome-mediated degradation. J Biol Chem 276:993–998

    Article  PubMed  CAS  Google Scholar 

  37. Vazquez F, Grossman SR, Takahashi Y, Rokas MV, Nakamura N, Sellers WR (2001) Phosphorylation of the PTEN tail acts as an inhibitory switch by preventing its recruitment into a protein complex. J Biol Chem 276:48627–48630

    Article  PubMed  CAS  Google Scholar 

  38. Rapkiewicz A, Espina V, Zujewski Ja, Lebowitz PF, Filie A, Wulfkuhle J, Camphausen K, Petricoin EF III, Liotta LA, Abati A (2007) The needle in the haystack: application of breast fine-needle aspirate samples to quantitative protein microarray technology. Cancer 111:173–184

    Article  PubMed  CAS  Google Scholar 

  39. Ahmad KA, Wang G, Unger G, Slaton J, Ahmed K (2008) Protein kinase CK2—a key suppressor of apoptosis. Adv Enzymol Regul 48:179–187

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The authors thank Susan G. Komen for the Cure foundation for financial support to Drs. Yu and Seewaldt (KG091020) and Dr. Ibarra-Drendall (KG090730). Dr. Seewaldt is also supported by the National Institute of Health/National Cancer Institute (R01CA88799 and R01CA114068). Additional financial support was provided by NCI/AVON Partners in Progress Grant 3 P30 CA014236-32S1 to Drs. Seewaldt and Yu.

Author information

Authors and Affiliations

  1. Division of Medical Oncology, Duke University Medical Center, Box 2628, Durham, NC, 27710, USA

    Catherine Ibarra-Drendall, Michelle M. Troch, Siya Lem, Joseph C. Baker Jr., Nicole M. Kuderer, Abigail W. Hoffman, Melanie Shivraj, Priya Mehta, Jamila Williams, Nora Tolbert, Laurie W. Lee, Patrick G. Pilie & Victoria L. Seewaldt

  2. Department of Biostatistics & Bioinformatics, Duke University Medical Center, Durham, NC, 27710, USA

    William T. Barry & Gloria Broadwater

  3. Center for Applied Proteomics and Molecular Medicine, George Mason University, Manassas, VA, 20110, USA

    Emanuel F. Petricoin III, Julia Wulfkuhle & Lance A. Liotta

  4. Department of Obstetrics and Gynecology, Duke University Medical Center, Durham, NC, 27710, USA

    Anne C. Ford

  5. Division of General Surgery, University of Wisconsin, Madison, WI, 53792, USA

    Lee G. Wilke

  6. Texas A&M Health Sciences Center, College of Medicine, Round Rock, TX, 78665, USA

    Carola Zalles

  7. Department of Molecular and Cellular Oncology, MD Anderson Cancer Center, Houston, TX, 77030, USA

    Dihua Yu

Authors
  1. Catherine Ibarra-Drendall
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Michelle M. Troch
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. William T. Barry
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Gloria Broadwater
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Emanuel F. Petricoin III
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Julia Wulfkuhle
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Lance A. Liotta
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Siya Lem
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Joseph C. Baker Jr.
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Anne C. Ford
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Lee G. Wilke
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Carola Zalles
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Nicole M. Kuderer
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Abigail W. Hoffman
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Melanie Shivraj
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Priya Mehta
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Jamila Williams
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. Nora Tolbert
    View author publications

    You can also search for this author in PubMed Google Scholar

  19. Laurie W. Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  20. Patrick G. Pilie
    View author publications

    You can also search for this author in PubMed Google Scholar

  21. Dihua Yu
    View author publications

    You can also search for this author in PubMed Google Scholar

  22. Victoria L. Seewaldt
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Catherine Ibarra-Drendall.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ibarra-Drendall, C., Troch, M.M., Barry, W.T. et al. Pilot and feasibility study: prospective proteomic profiling of mammary epithelial cells from high-risk women provides evidence of activation of pro-survival pathways. Breast Cancer Res Treat 132, 487–498 (2012). https://doi.org/10.1007/s10549-011-1609-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10549-011-1609-9

Keywords

Search

Navigation