Article | Published:

C/EBPδ links IL-6 and HIF-1 signaling to promote breast cancer stem cell-associated phenotypes

Oncogene (2018) | Download Citation


To improve cancer patient outcome significantly, we must understand the mechanisms regulating stem-like cancer cells, which have been implicated as a cause of metastasis and treatment resistance. The transcription factor C/EBPδ can exhibit pro- and anti-tumorigenic activities, but the mechanisms underlying the complexity of its functions are poorly understood. Here we identify a role for breast cancer cell intrinsic C/EBPδ in promoting phenotypes that have been associated with cancer stem cells (CSCs). While C/EBPδ expression is not abundant in most metastatic breast cancers, our data support a pro-tumorigenic role of C/EBPδ when expressed in subsets of tumor cells and/or through transient activation by the tumor microenvironment or loss of substrate adhesion. Using genetic mouse models and human breast cancer cell lines, we show that deletion or depletion of C/EBPδ reduced expression of stem cell factors and stemnness markers, sphere formation and self-renewal, along with growth of tumors and established experimental metastases in vivo. C/EBPδ is also known as a mediator of the innate immune response, which is enhanced by hypoxia and interleukin-6 (IL-6) signaling, two conditions that also play important roles in cancer progression. Our mechanistic data reveal C/EBPδ as a link that engages two positive feedback loops, in part by directly targeting the IL-6 receptor (IL6RA) gene, and, thus, amplifying IL-6 and HIF-1 signaling. This study provides a molecular mechanism for the synergism of tumor microenvironmental conditions in cancer progression with potential implications for the targeting of CSCs.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


  1. 1.

    Tabassum DP, Polyak K. Tumorigenesis: it takes a village. Nat Rev Cancer. 2015;15:473–83.

  2. 2.

    Valent P, Bonnet D, De Maria R, Lapidot T, Copland M, Melo JV, et al. Cancer stem cell definitions and terminology: the devil is in the details. Nat Rev Cancer. 2012;12:767–75.

  3. 3.

    Wei W, Lewis MT. Identifying and targeting tumor-initiating cells in the treatment of breast cancer. Endocr Relat Cancer. 2015;22:R135–155.

  4. 4.

    Brower V.. Cancer stem cell hypothesis evolves with emerging research. J Natl Cancer Inst. 2016;108:2–4.

  5. 5.

    Espinoza I, Pochampally R, Xing F, Watabe K, Miele L. Notch signaling: targeting cancer stem cells and epithelial-to-mesenchymal transition. Onco Targets Ther. 2013;6:1249–59.

  6. 6.

    Friedmann-Morvinski D, Verma IM. Dedifferentiation and reprogramming: origins of cancer stem cells. EMBO Rep. 2014;15:244–53.

  7. 7.

    Visvader JE, Lindeman GJ. Cancer stem cells: current status and evolving complexities. Cell Stem Cell. 2012;10:717–28.

  8. 8.

    Balamurugan K. HIF-1 at the crossroads of hypoxia, inflammation, and cancer. Int J Cancer. 2016;138:1058–66.

  9. 9.

    Korkaya H, Liu S, Wicha MS. Regulation of cancer stem cells by cytokine networks: attacking cancer’s inflammatory roots. Clin Cancer Res. 2011;17:6125–9.

  10. 10.

    Mathieu J, Zhang Z, Zhou W, Wang AJ, Heddleston JM, Pinna CM, et al. HIF induces human embryonic stem cell markers in cancer cells. Cancer Res. 2011;71:4640–52.

  11. 11.

    Chang Q, Daly L, Bromberg J. The IL-6 feed-forward loop: a driver of tumorigenesis. Semin Immunol. 2014;26:48–53.

  12. 12.

    Marotta LL, Almendro V, Marusyk A, Shipitsin M, Schemme J, Walker SR, et al. The JAK2/STAT3 signaling pathway is required for growth of CD44( + )CD24(-) stem cell-like breast cancer cells in human tumors. J Clin Invest. 2011;121:2723–35.

  13. 13.

    Taniguchi K, Karin M. IL-6 and related cytokines as the critical lynchpins between inflammation and cancer. Semin Immunol. 2014;26:54–74.

  14. 14.

    Wei W, Tweardy DJ, Zhang M, Zhang X, Landua J, Petrovic I, et al. STAT3 signaling is activated preferentially in tumor-initiating cells in claudin-low models of human breast cancer. Stem Cells. 2014;32:2571–82.

  15. 15.

    Balamurugan K, Sterneck E. The many faces of C/EBPdelta and their implications in inflammation and cancer. Int J Biol Sci. 2013;9:917–33.

  16. 16.

    Balamurugan K, Wang JM, Tsai HH, Sharan S, Anver M, Leighty R, et al. The tumour suppressor C/EBPdelta inhibits FBXW7 expression and promotes mammary tumour metastasis. EMBO J. 2010;29:4106–17.

  17. 17.

    Yamaguchi J, Tanaka T, Eto N, Nangaku M. Inflammation and hypoxia linked to renal injury by CCAAT/enhancer-binding protein delta. Kidney Int. 2015;88:262–75.

  18. 18.

    Litvak V, Ramsey SA, Rust AG, Zak DE, Kennedy KA, Lampano AE, et al. Function of C/EBPdelta in a regulatory circuit that discriminates between transient and persistent TLR4-induced signals. Nat Immunol. 2009;10:437–43.

  19. 19.

    Wang YH, Wu WJ, Wang WJ, Huang HY, Li WM, Yeh BW. CEBPD amplification and over expression in urothelial carcinoma: a driver of tumor metastasis indicating adverse prognosis. Oncotarget. 2015;6:31069–84.

  20. 20.

    Chen JC, Alvarez MJ, Talos F, Dhruv H, Rieckhof GE, Iyer A, et al. Identification of causal genetic drivers of human disease through systems-level analysis of regulatory networks. Cell. 2014;159:402–14.

  21. 21.

    Min Y, Ghose S, Boelte K, Li J, Yang L, Lin PC. C/EBP-delta regulates VEGF-C autocrine signaling in lymphangiogenesis and metastasis of lung cancer through HIF-1alpha. Oncogene. 2011;30:4901–9.

  22. 22.

    Min Y, Li J, Qu P, Lin PC. C/EBP-delta positively regulates MDSC expansion and endothelial VEGFR2 expression in tumor development. Oncotarget. 2017;8:50582–93.

  23. 23.

    Mitra A, Mishra L, Li S. EMT, CTCs and CSCs in tumor relapse and drug-resistance. Oncotarget. 2015;6:10697–711.

  24. 24.

    Lo PK, Kanojia D, Liu X, Singh UP, Berger FG, Wang Q, et al. CD49f and CD61 identify Her2/neu-induced mammary tumor-initiating cells that are potentially derived from luminal progenitors and maintained by the integrin-TGFbeta signaling. Oncogene. 2012;31:2614–26.

  25. 25.

    Sarkar TR, Sharan S, Wang J, Pawar SA, Cantwell CA, Johnson PF, et al. Identification of a Src tyrosine kinase/SIAH2 E3 ubiquitin ligase pathway that regulates C/EBPdelta expression and contributes to transformation of breast tumor cells. Mol Cell Biol. 2012;32:320–32.

  26. 26.

    Prat A, Parker JS, Karginova O, Fan C, Livasy C, Herschkowitz JI, et al. Phenotypic and molecular characterization of the claudin-low intrinsic subtype of breast cancer. Breast Cancer Res. 2010;12:R68.

  27. 27.

    Lagadec C, Vlashi E, Frohnen P, Alhiyari Y, Chan M, Pajonk F. The RNA-binding protein Musashi-1 regulates proteasome subunit expression in breast cancer- and glioma-initiating cells. Stem Cells. 2014;32:135–44.

  28. 28.

    Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF. Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci USA. 2003;100:3983–8.

  29. 29.

    Mendoza-Villanueva D, Balamurugan K, Ali HR, Kim SR, Sharan S, Johnson RC. The C/EBPdelta protein is stabilized by estrogen receptor alpha activity, inhibits SNAI2 expression and associates with good prognosis in breast cancer. Oncogene. 2016;35:6166–76.

  30. 30.

    Harrison H, Rogerson L, Gregson HJ, Brennan KR, Clarke RB, Landberg G. Contrasting hypoxic effects on breast cancer stem cell hierarchy is dependent on ER-alpha status. Cancer Res. 2013;73:1420–33.

  31. 31.

    Sansone P, Storci G, Tavolari S, Guarnieri T, Giovannini C, Taffurelli M, et al. IL-6 triggers malignant features in mammospheres from human ductal breast carcinoma and normal mammary gland. J Clin Invest. 2007;117:3988–4002.

  32. 32.

    Yan W, Chen Y, Yao Y, Zhang H, Wang T. Increased invasion and tumorigenicity capacity of CD44 + /CD24- breast cancer MCF7 cells in vitro and in nude mice. Cancer Cell Int. 2013;13:62.

  33. 33.

    Mihara M, Hashizume M, Yoshida H, Suzuki M, Shiina M. IL-6/IL-6 receptor system and its role in physiological and pathological conditions. Clin Sci (Lond). 2012;122:143–59.

  34. 34.

    Balamurugan K, Sharan S, Klarmann KD, Zhang Y, Coppola V, Summers GH, et al. FBXW7alpha attenuates inflammatory signalling by downregulating C/EBPdelta and its target gene Tlr4. Nat Commun. 2013;4:1662.

  35. 35.

    Zou J, Li P, Lu F, Liu N, Dai J, Ye J, et al. Notch1 is required for hypoxia-induced proliferation, invasion and chemoresistance of T-cell acute lymphoblastic leukemia cells. J Hematol Oncol. 2013;6:3.

  36. 36.

    Kageyama Y, Koshiji M, To KK, Tian YM, Ratcliffe PJ, Huang LE. Leu-574 of human HIF-1alpha is a molecular determinant of prolyl hydroxylation. FASEB J. 2004;18:1028–30.

  37. 37.

    Davis RJ, Welcker M, Clurman BE. Tumor suppression by the Fbw7 ubiquitin ligase: mechanisms and opportunities. Cancer Cell. 2014;26:455–64.

  38. 38.

    Zhang X, Claerhout S, Prat A, Dobrolecki LE, Petrovic I, Lai Q, et al. A renewable tissue resource of phenotypically stable, biologically and ethnically diverse, patient-derived human breast cancer xenograft models. Cancer Res. 2013;73:4885–97.

  39. 39.

    Gwak JM, Kim M, Kim HJ, Jang MH, Park SY. Expression of embryonal stem cell transcription factors in breast cancer: Oct4 as an indicator for poor clinical outcome and tamoxifen resistance. Oncotarget. 2017;8:36305–18.

  40. 40.

    Fallah Y, Brundage J, Allegakoen P, Shajahan-Haq AN. MYC-driven pathways in breast cancer subtypes. Biomolecules. 2017;7:53–58.

  41. 41.

    Yang A, Qin S, Schulte BA, Ethier SP, Tew KD, Wang GY. MYC inhibition depletes cancer stem-like cells in triple-negative breast cancer. Cancer Res. 2017;77:6641–50.

  42. 42.

    Cojoc M, Peitzsch C, Trautmann F, Polishchuk L, Telegeev GD, Dubrovska A. Emerging targets in cancer management: role of the CXCL12/CXCR4 axis. Onco Targets Ther. 2013;6:1347–61.

  43. 43.

    Rustighi A, Zannini A, Tiberi L, Sommaggio R, Piazza S, Sorrentino G, et al. Prolyl-isomerase Pin1 controls normal and cancer stem cells of the breast. EMBO Mol Med. 2014;6:99–119.

  44. 44.

    Mamlouk S, Wielockx B. Hypoxia-inducible factors as key regulators of tumor inflammation. Int J Cancer. 2013;132:2721–9.

  45. 45.

    Gustafsson MV, Zheng X, Pereira T, Gradin K, Jin S, Lundkvist J, et al. Hypoxia requires notch signaling to maintain the undifferentiated cell state. Dev Cell. 2005;9:617–28.

  46. 46.

    Seymour T, Twigger AJ, Kakulas F. Pluripotency genes and their functions in the normal and aberrant breast and brain. Int J Mol Sci. 2015;16:27288–301.

  47. 47.

    Dobrolecki LE, Airhart SD, Alferez DG, Aparicio S, Behbod F, Bentires-Alj M, et al. Patient-derived xenograft (PDX) models in basic and translational breast cancer research. Cancer Metastasis Rev. 2016;35:547–73.

  48. 48.

    Luo M, Brooks M, Wicha MS. Epithelial-mesenchymal plasticity of breast cancer stem cells: implications for metastasis and therapeutic resistance. Curr Pharm Des. 2015;21:1301–10.

  49. 49.

    Watanabe M, Uehara Y, Yamashita N, Fujimura Y, Nishio K, Sawada T, et al. Multicolor detection of rare tumor cells in blood using a novel flow cytometry-based system. Cytometry A. 2014;85:206–13.

Download references


We are thankful for superb support through services provided by Leidos Biomedical Research, Inc., FNLCR (Laboratory Animal Sciences Program, Protein Expression Laboratory, Illustrations, and Graphical Support); and the NCI/CCR cores (Small Animal Imaging, Flow Cytometry, Optical Microscopy, and Analysis Laboratory); and Data Management Services, Inc. for assistance with Statistics. We thank Linda Miller, Suzanne Specht, Rena Mao, and the student interns for their valuable contributions; Heidi Dowst for providing clinical information related to the PDX models; and all the investigators who kindly shared their valuable reagents (see Methods) or provided critical comments on the manuscript. This research was supported by the Intramural Research Program of the NIH, National Cancer Institute, in part with Federal Funds from the Frederick Cancer Institute (NIH) under contract no. HHSN261200800001E. LED and MTL were supported by BCRF Founders Fund Grant CPRIT DP150069 and V-Foundation grant T2014-010. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.

Author contributions

KB and ES conceived the project and designed the study. KB, DM-V, SS, and ES designed, conducted, and/or analyzed the experiments. LED and MTL provided PDX reagents, data, resources, and advice. GHS and LED provided technical support and supervision for animal model studies. KB, MTL, and ES wrote the manuscript.

Author information

Author notes

    • Daniel Mendoza-Villanueva

    Present address: Mission Bio, South San Francisco, CA, 94080, USA


  1. Laboratory of Cell and Developmental Signaling, Center for Cancer Research, National Cancer Institute, Frederick, MD, 21702, USA

    • Kuppusamy Balamurugan
    • , Daniel Mendoza-Villanueva
    • , Shikha Sharan
    •  & Esta Sterneck
  2. Laboratory Animal Sciences Program, Leidos Biomedical Research Inc., Frederick, MD, 21702, USA

    • Glenn H. Summers
  3. Departments of Molecular and Cellular Biology and Radiology, Lester and Sue Smith Breast Center, Baylor College of Medicine, Houston, TX, 77030, USA

    • Lacey E. Dobrolecki
    •  & Michael T. Lewis


  1. Search for Kuppusamy Balamurugan in:

  2. Search for Daniel Mendoza-Villanueva in:

  3. Search for Shikha Sharan in:

  4. Search for Glenn H. Summers in:

  5. Search for Lacey E. Dobrolecki in:

  6. Search for Michael T. Lewis in:

  7. Search for Esta Sterneck in:

Conflict of interest

MTL is a limited partner in StemMed Ltd, and a Manager in StemMed Holdings, its general partner. He also holds an equity stake in Tvardi Therapeutics Inc. LED is a compensated employee of StemMed Ltd. The remaining authors declare that they have no conflict of interests.

Corresponding authors

Correspondence to Kuppusamy Balamurugan or Esta Sterneck.

Electronic supplementary material

About this article

Publication history