Skip to main content

Advertisement

SpringerLink
Log in

HITS-CLIP reveals key regulators of nuclear receptor signaling in breast cancer

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

Abstract

miRNAs regulate the expression of genes in both normal physiology and disease. While miRNAs have been demonstrated to play a pivotal role in aspects of cancer biology, these reports have generally focused on the regulation of single genes. Such single-gene approaches have significant limitations, relying on miRNA expression levels and heuristic predictions of mRNA-binding sites. This results in only circumstantial evidence of miRNA–target interaction and typically leads to large numbers of false positive predictions. Here, we used a genome-wide approach (high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation, HITS-CLIP) to define direct miRNA–mRNA interactions in three breast cancer subtypes (estrogen receptor positive, Her2 amplified, and triple negative). Focusing on steroid receptor signaling, we identified two novel regulators of the ER pathway (miR-9-5p and miR-193a/b-3p), which together target multiple genes involved in ER signaling. Moreover, this approach enabled the definition of miR-9-5p as a global regulator of steroid receptor signaling in breast cancer. We show that miRNA targets and networks defined by HITS-CLIP under physiologic conditions are predictive of patient outcomes and provide global insight into miRNA regulation in breast cancer.

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
Fig. 6

Similar content being viewed by others

References

  1. Desantis C, Ma J, Bryan L, Jemal A (2013) Breast cancer statistics, 2013. CA Cancer J Clin. doi:10.3322/caac.21203

    Google Scholar 

  2. Musgrove EA, Sutherland RL (2009) Biological determinants of endocrine resistance in breast cancer. Nat Rev Cancer 9:631–643. doi:10.1038/nrc2713

    Article  CAS  PubMed  Google Scholar 

  3. Croce CM (2009) Causes and consequences of microRNA dysregulation in cancer. Nat Rev Genet 10:704–714. doi:10.1038/nrg2634

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  4. He L, Hannon GJ (2004) MicroRNAs: small RNAs with a big role in gene regulation. Nat Rev Genet 5:522–531. doi:10.1038/nrg1379

    Article  CAS  PubMed  Google Scholar 

  5. Li H, Bian C, Liao L, Li J, Zhao RC (2010) miR-17-5p promotes human breast cancer cell migration and invasion through suppression of HBP1. Breast Cancer Res Treat. doi:10.1007/s10549-010-0954-4

    Google Scholar 

  6. Zhao J-J, Lin J, Yang H, Kong W, He L, Ma X, Coppola D, Cheng JQ (2008) MicroRNA-221/222 negatively regulates estrogen receptor alpha and is associated with tamoxifen resistance in breast cancer. J Biol Chem 283:31079–31086. doi:10.1074/jbc.M806041200

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  7. Thomson DW, Bracken CP, Goodall GJ (2011) Experimental strategies for microRNA target identification. Nucleic Acids Res 39:6845–6853. doi:10.1093/nar/gkr330

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  8. Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116:281–297

    Article  CAS  PubMed  Google Scholar 

  9. Rajewsky N (2006) microRNA target predictions in animals. Nat Genet 38(Suppl):S8–S13. doi:10.1038/ng1798

    Article  CAS  PubMed  Google Scholar 

  10. Chi SW, Zang JB, Mele A, Darnell RB (2009) Argonaute HITS-CLIP decodes microRNA–mRNA interaction maps. Nature 460:479–486. doi:10.1038/nature08170

    CAS  PubMed Central  PubMed  Google Scholar 

  11. Licatalosi DD, Mele A, Fak JJ, Ule J, Kayikci M, Chi SW, Clark TA, Schweitzer AC, Blume JE, Wang X, Darnell JC, Darnell RB (2008) HITS-CLIP yields genome-wide insights into brain alternative RNA processing. Nature 456:464–469. doi:10.1038/nature07488

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  12. Keydar I, Chen L, Karby S, Weiss FR, Delarea J, Radu M, Chaitcik S, Brenner HJ (1979) Establishment and characterization of a cell line of human breast carcinoma origin. Eur J Cancer 15:659–670

    Article  CAS  PubMed  Google Scholar 

  13. Korch C, Spillman MA, Jackson TA, Jacobsen BM, Murphy SK, Lessey BA, Jordan VC, Bradford AP (2012) DNA profiling analysis of endometrial and ovarian cell lines reveals misidentification, redundancy and contamination. Gynecol Oncol 127:241–248. doi:10.1016/j.ygyno.2012.06.017

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  14. Pillai MM, Yang X, Balakrishnan I, Bemis L, Torok-Storb B (2010) MiR-886-3p down regulates CXCL12 (SDF1) expression in human marrow stromal cells. PLoS One 5:e14304. doi:10.1371/journal.pone.0014304

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  15. Chen C-Z, Li L, Lodish HF, Bartel DP (2004) MicroRNAs modulate hematopoietic lineage differentiation. Science 303:83–86. doi:10.1126/science.1091903

    Article  CAS  PubMed  Google Scholar 

  16. Balakrishnan I, Yang X, Brown J, Ramakrishnan A, Torok-Storb B, Kabos P, Hesselberth JR, Pillai MM (2013) Genome-wide analysis of miRNA–mRNA interactions in marrow stromal cells. Stem Cells. doi:10.1002/stem.1531

    Google Scholar 

  17. Niu G, Wright KL, Huang M, Song L, Haura E, Turkson J, Zhang S, Wang T, Sinibaldi D, Coppola D, Heller R, Ellis LM, Karras J, Bromberg J, Pardoll D, Jove R, Yu H (2002) Constitutive Stat3 activity up-regulates VEGF expression and tumor angiogenesis. Oncogene 21:2000–2008. doi:10.1038/sj.onc.1205260

    Article  CAS  PubMed  Google Scholar 

  18. Yeo GW, Coufal NG, Liang TY, Peng GE, Fu X-D, Gage FH (2009) An RNA code for the FOX2 splicing regulator revealed by mapping RNA–protein interactions in stem cells. Nat Struct Mol Biol 16:130–137. doi:10.1038/nsmb.1545

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  19. Buffa FM, Camps C, Winchester L, Snell CE, Gee HE, Sheldon H, Taylor M, Harris AL, Ragoussis J (2011) microRNA-associated progression pathways and potential therapeutic targets identified by integrated mRNA and microRNA expression profiling in breast cancer. Cancer Res 71:5635–5645. doi:10.1158/0008-5472.CAN-11-0489

    Article  CAS  PubMed  Google Scholar 

  20. Cancer Genome Atlas Network (2012) Comprehensive molecular portraits of human breast tumours. Nature 490:61–70. doi:10.1038/nature11412

    Article  Google Scholar 

  21. Neve RM, Chin K, Fridlyand J, Yeh J, Baehner FL, Fevr T, Clark L, Bayani N, Coppe J-P, Tong F, Speed T, Spellman PT, DeVries S, Lapuk A, Wang NJ, Kuo W-L, Stilwell JL, Pinkel D, Albertson DG, Waldman FM, McCormick F, Dickson RB, Johnson MD, Lippman M, Ethier S, Gazdar A, Gray JW (2006) A collection of breast cancer cell lines for the study of functionally distinct cancer subtypes. Cancer Cell 10:515–527. doi:10.1016/j.ccr.2006.10.008

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  22. Darnell RB (2010) HITS-CLIP: panoramic views of protein–RNA regulation in living cells. Wiley Interdiscip Rev RNA 1:266–286. doi:10.1002/wrna.31

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  23. Wang Z, Gerstein M, Snyder M (2009) RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet 10:57–63. doi:10.1038/nrg2484

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  24. Hurteau GJ, Carlson JA, Spivack SD, Brock GJ (2007) Overexpression of the microRNA hsa-miR-200c leads to reduced expression of transcription factor 8 and increased expression of E-cadherin. Cancer Res 67:7972–7976. doi:10.1158/0008-5472.CAN-07-1058

    Article  CAS  PubMed  Google Scholar 

  25. Stinson S, Lackner MR, Adai AT, Yu N, Kim H-J, O’Brien C, Spoerke J, Jhunjhunwala S, Boyd Z, Januario T, Newman RJ, Yue P, Bourgon R, Modrusan Z, Stern HM, Warming S, de Sauvage FJ, Amler L, Yeh R-F, Dornan D (2011) TRPS1 targeting by miR-221/222 promotes the epithelial-to-mesenchymal transition in breast cancer. Sci Signal 4:ra41. doi:10.1126/scisignal.2001538

  26. Lopes CT, Franz M, Kazi F, Donaldson SL, Morris Q, Bader GD (2010) Cytoscape web: an interactive web-based network browser. Bioinforma Oxf Engl 26:2347–2348. doi:10.1093/bioinformatics/btq430

    Article  CAS  Google Scholar 

  27. Avery-Kiejda KA, Braye SG, Forbes JF, Scott RJ (2014) The expression of Dicer and Drosha in matched normal tissues, tumours and lymph node metastases in triple negative breast cancer. BMC Cancer 14:253. doi:10.1186/1471-2407-14-253

    Article  PubMed Central  PubMed  Google Scholar 

  28. Di Leva G, Gasparini P, Piovan C, Ngankeu A, Garofalo M, Taccioli C, Iorio MV, Li M, Volinia S, Alder H, Nakamura T, Nuovo G, Liu Y, Nephew KP, Croce CM (2010) MicroRNA cluster 221–222 and estrogen receptor alpha interactions in breast cancer. J Natl Cancer Inst 102:706–721. doi:10.1093/jnci/djq102

    Article  PubMed Central  PubMed  Google Scholar 

  29. Castellano L, Giamas G, Jacob J, Coombes RC, Lucchesi W, Thiruchelvam P, Barton G, Jiao LR, Wait R, Waxman J, Hannon GJ, Stebbing J (2009) The estrogen receptor-α-induced microRNA signature regulates itself and its transcriptional response. Proc Natl Acad Sci 106:15732–15737. doi:10.1073/pnas.0906947106

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  30. Chen L, Zheng J, Zhang Y, Yang L, Wang J, Ni J, Cui D, Yu C, Cai Z (2011) Tumor-specific expression of microRNA-26a suppresses human hepatocellular carcinoma growth via cyclin-dependent and -independent pathways. Mol Ther J Am Soc Gene Ther 19:1521–1528. doi:10.1038/mt.2011.64

    Article  CAS  Google Scholar 

  31. Keaveney M, Parker MG, Gannon F (1993) Identification of a functional role for the 3′ region of the human oestrogen receptor gene. J Mol Endocrinol 10:143–152

    Article  CAS  PubMed  Google Scholar 

  32. Miranda KC, Huynh T, Tay Y, Ang Y-S, Tam W-L, Thomson AM, Lim B, Rigoutsos I (2006) A pattern-based method for the identification of microRNA binding sites and their corresponding heteroduplexes. Cell 126:1203–1217. doi:10.1016/j.cell.2006.07.031

    Article  CAS  PubMed  Google Scholar 

  33. Rehmsmeier M, Steffen P, Hochsmann M, Giegerich R (2004) Fast and effective prediction of microRNA/target duplexes. RNA 10:1507–1517. doi:10.1261/rna.5248604

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  34. Buache E, Etique N, Alpy F, Stoll I, Muckensturm M, Reina-San-Martin B, Chenard MP, Tomasetto C, Rio MC (2011) Deficiency in trefoil factor 1 (TFF1) increases tumorigenicity of human breast cancer cells and mammary tumor development in TFF1-knockout mice. Oncogene 30:3261–3273. doi:10.1038/onc.2011.41

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  35. Foekens JA, Rio MC, Seguin P, van Putten WL, Fauque J, Nap M, Klijn JG, Chambon P (1990) Prediction of relapse and survival in breast cancer patients by pS2 protein status. Cancer Res 50:3832–3837

    CAS  PubMed  Google Scholar 

  36. Clark GM, McGuire WL, Hubay CA, Pearson OH, Marshall JS (1983) Progesterone receptors as a prognostic factor in stage II breast cancer. N Engl J Med 309:1343–1347

    Article  CAS  PubMed  Google Scholar 

  37. Jordan VC, O’Malley BW (2007) Selective estrogen-receptor modulators and antihormonal resistance in breast cancer. J Clin Oncol 25:5815–5824. doi:10.1200/JCO.2007.11.3886

    Article  CAS  PubMed  Google Scholar 

  38. Osborne CK, Bardou V, Hopp TA, Chamness GC, Hilsenbeck SG, Fuqua SAW, Wong J, Allred DC, Clark GM, Schiff R (2003) Role of the estrogen receptor coactivator AIB1 (SRC-3) and HER-2/neu in tamoxifen resistance in breast cancer. J Natl Cancer Inst 95:353–361

    Article  CAS  PubMed  Google Scholar 

  39. Arpino G, Wiechmann L, Osborne CK, Schiff R (2008) Crosstalk between the estrogen receptor and the HER tyrosine kinase receptor family: molecular mechanism and clinical implications for endocrine therapy resistance. Endocr Rev 29:217–233. doi:10.1210/er.2006-0045

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  40. Osborne CK, Schiff R (2011) Mechanisms of endocrine resistance in breast cancer. Annu Rev Med 62:233–247. doi:10.1146/annurev-med-070909-182917

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  41. Papai G, Weil PA, Schultz P (2011) New insights into the function of transcription factor TFIID from recent structural studies. Curr Opin Genet Dev 21:219–224. doi:10.1016/j.gde.2011.01.009

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  42. Gasparini P, Fassan M, Cascione L, Guler G, Balci S, Irkkan C, Paisie C, Lovat F, Morrison C, Zhang J, Scarpa A, Croce CM, Shapiro CL, Huebner K (2014) Androgen receptor status is a prognostic marker in non-basal triple negative breast cancers and determines novel therapeutic options. PLoS One 9:e88525. doi:10.1371/journal.pone.0088525

    Article  PubMed Central  PubMed  Google Scholar 

  43. Park S, Park HS, Koo JS, Yang WI, Kim SI, Park B-W (2012) Higher expression of androgen receptor is a significant predictor for better endocrine-responsiveness in estrogen receptor-positive breast cancers. Breast Cancer Res Treat 133:311–320. doi:10.1007/s10549-011-1950-z

    Article  CAS  PubMed  Google Scholar 

  44. Peters KM, Edwards SL, Nair SS, French JD, Bailey PJ, Salkield K, Stein S, Wagner S, Francis GD, Clark SJ, Brown MA (2012) Androgen receptor expression predicts breast cancer survival: the role of genetic and epigenetic events. BMC Cancer 12:132. doi:10.1186/1471-2407-12-132

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  45. Perou CM, Sørlie T, Eisen MB, van de Rijn M, Jeffrey SS, Rees CA, Pollack JR, Ross DT, Johnsen H, Akslen LA, Fluge O, Pergamenschikov A, Williams C, Zhu SX, Lønning PE, Børresen-Dale AL, Brown PO, Botstein D (2000) Molecular portraits of human breast tumours. Nature 406:747–752. doi:10.1038/35021093

    Article  CAS  PubMed  Google Scholar 

  46. Helwak A, Kudla G, Dudnakova T, Tollervey D (2013) Mapping the human miRNA interactome by CLASH reveals frequent noncanonical binding. Cell 153:654–665. doi:10.1016/j.cell.2013.03.043

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  47. Kivioja T, Vaharautio A, Karlsson K, Bonke M, Enge M, Linnarsson S, Taipale J (2012) Counting absolute numbers of molecules using unique molecular identifiers. Nat Methods 9:72–74. doi:10.1038/nmeth.1778

    CAS  Google Scholar 

  48. Kishore S, Jaskiewicz L, Burger L, Hausser J, Khorshid M, Zavolan M (2011) A quantitative analysis of CLIP methods for identifying binding sites of RNA-binding proteins. Nat Methods 8:559–564. doi:10.1038/nmeth.1608

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

We are thankful to Dr. Zissimos Mourelatos (University of Pennsylvania, Philadelphia, PA) for generously providing the Ago antibody for initial experiments, Jill Castoe (High-Throughput Sequencing Core, UC Denver) for assistance with next generation sequencing, Dr. Carol Sartorius (UC Denver) for critical reading of the manuscript, and Dr. Kenneth Jones (UC Denver) for logistical support of data analysis. This study was supported in part by Grants from the National Institute of Health (HL104070 and DK073701) to MMP and CA164048 to PK; Grohne Cancer Research Fund to PK, March of Dimes Basil O’Connor Starter Scholar, Damon Runyon Rachleff Innovation Award and American Cancer Society Research Scholar Grant to J.R.H.

Conflict of interest

The authors declare no conflicts of interest.

Author information

Authors and Affiliations

  1. Section of Hematology, Yale Cancer Center, New Haven, CT, USA

    Manoj M. Pillai

  2. Department of Medicine, University of Colorado Denver, 12801 E 17th Ave., L18-4101, MS 8117, Aurora, CO, USA

    Austin E. Gillen, Tomomi M. Yamamoto, Enos Kline, Kale Flory & Peter Kabos

  3. Department of Biochemistry and Molecular Genetics, University of Colorado Denver, Aurora, CO, USA

    Austin E. Gillen, Joseph Brown & Jay R. Hesselberth

Authors
  1. Manoj M. Pillai
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Austin E. Gillen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Tomomi M. Yamamoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Enos Kline
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Joseph Brown
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Kale Flory
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jay R. Hesselberth
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Peter Kabos
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Peter Kabos.

Additional information

Manoj M. Pillai and Austin E. Gillen contributed equally to this work.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 701 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Pillai, M.M., Gillen, A.E., Yamamoto, T.M. et al. HITS-CLIP reveals key regulators of nuclear receptor signaling in breast cancer. Breast Cancer Res Treat 146, 85–97 (2014). https://doi.org/10.1007/s10549-014-3004-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10549-014-3004-9

Keywords

Search

Navigation