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Interaction between STAT3 and GLI1/tGLI1 oncogenic transcription factors promotes the aggressiveness of triple-negative breast cancers and HER2-enriched breast cancer

Oncogenevolume 37pages25022514 (2018) | Download Citation


Signal transducer and activator of transcription 3 (STAT3), glioma oncogene homolog 1 (GLI1), and truncated GLI1 (tGLI1) are oncogenic transcription factors playing important roles in breast cancer. tGLI1 is a gain-of-function GLI1 isoform. Whether STAT3 physically and/or functionally interacts with GLI1/tGLI1 has not been explored. To address this knowledge gap, we analyzed 47 node-positive breast cancer specimens using immunohistochemical staining and found that phosphorylated-STAT3 (Y705), GLI1, and tGLI1 are co-overexpressed in the majority of triple-negative breast carcinomas (64%) and HER2-enriched (68%) breast carcinomas, and in lymph node metastases (65%). Using gene set enrichment analysis, we analyzed 710 breast tumors and found that STAT3 activation and GLI1/tGLI1 activation signatures are co-enriched in triple-negative subtypes of breast cancers and HER2-enriched subtypes of breast cancers, but not in luminal subtypes of breast cancers. Patients with high levels of STAT3 and GLI1/tGLI1 co-activation in their breast tumors had worse metastasis-free survival compared to those with low levels. Since these proteins co-overexpress in breast tumors, we examined whether they form complexes and observed that STAT3 interacted with both GLI1 and tGLI1. We further found that the STAT3-GLI1 and STAT3-tGLI1 complexes bind to both consensus GLI1-binding and STAT3-binding sites using chromatin immunoprecipitation (ChIP) assay, and that the co-overexpression markedly activated a promoter controlled by GLI1-binding sites. To identify genes that can be directly co-activated by STAT3 and GLI1/tGLI1, we analyzed three ChIP-seq datasets and identified 34 potential target genes. Following validations using reverse transcription polymerase chain reaction and survival analysis, we identified three genes as novel transcriptional targets of STAT3 and GLI1/tGLI1, R-Ras2, Cep70, and UPF3A. Finally, we observed that co-overexpression of STAT3 with GLI1/tGLI1 promoted the ability of breast cancer cells to form mammospheres and that STAT3 only cooperates with tGLI1 in immortalized mammary epithelial cells. In summary, our study identified novel physical and functional cooperation between two families of oncogenic transcription factors, and the interaction contributes to aggressiveness of breast cancer cells and poor prognosis of triple-negative breast cancers and HER2-enriched breast cancers.

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  1. 1.

    Cancer Genome Atlas N. Comprehensive molecular portraits of human breast tumours. Nature. 2012;490:61–70.

  2. 2.

    Abou-Bakr AA, Eldweny HI. p16 expression correlates with basal-like triple-negative breast carcinoma. Ecancermedicalscience. 2013;7:317.

  3. 3.

    De Brot M, Soares FA, Stiepcich MM, Curcio VS, Gobbi H. Basal-like breast cancers: clinicopathological features and outcome. Rev Assoc Med Bras. 2009;55:529–34.

  4. 4.

    Brouckaert O, Wildiers H, Floris G, Neven P. Update on triple-negative breast cancer: prognosis and management strategies. Int J Women’s Health. 2012;4:511–20.

  5. 5.

    Kuba S, Ishida M, Nakamura Y, Yamanouchi K, Minami S, Taguchi K, et al. Treatment and prognosis of breast cancer patients with brain metastases according to intrinsic subtype. Jpn J Clin Oncol. 2014;44:1025–31.

  6. 6.

    Paik S, Hazan R, Fisher ER, Sass RE, Fisher B, Redmond C, et al. Pathologic findings from the National Surgical Adjuvant Breast and Bowel Project: prognostic significance of erbB-2 protein overexpression in primary breast cancer. J Clin Oncol. 1990;8:103–12.

  7. 7.

    Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL. Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science. 1987;235:177–82.

  8. 8.

    Slamon DJ, Godolphin W, Jones LA, Holt JA, Wong SG, Keith DE, et al. Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science. 1989;244:707–12.

  9. 9.

    Esteva FJ, Valero V, Booser D, Guerra LT, Murray JL, Pusztai L, et al. Phase II study of weekly docetaxel and trastuzumab for patients with HER-2-overexpressing metastatic breast cancer. J Clin Oncol. 2002;20:1800–8.

  10. 10.

    Slamon DJ, Leyland-Jones B, Shak S, Fuchs H, Paton V, Bajamonde A, et al. Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N Engl J Med. 2001;344:783–92.

  11. 11.

    Geyer CE, Forster J, Lindquist D, Chan S, Romieu CG, Pienkowski T, et al. Lapatinib plus capecitabine for HER2-positive advanced breast cancer. N Engl J Med. 2006;355:2733–43.

  12. 12.

    Balko JM, Giltnane JM, Wang K, Schwarz LJ, Young CD, Cook RS, et al. Molecular profiling of the residual disease of triple-negative breast cancers after neoadjuvant chemotherapy identifies actionable therapeutic targets. Cancer Discov. 2014;4:232–45.

  13. 13.

    Balko JM, Schwarz LJ, Luo N, Estrada MV, Giltnane JM, Davila-Gonzalez D, et al. Triple-negative breast cancers with amplification of JAK2 at the 9p24 locus demonstrate JAK2-specific dependence. Sci Transl Med. 2016;8:334ra353.

  14. 14.

    Yu H, Jove R. The STATs of cancer—new molecular targets come of age. Nat Rev Cancer. 2004;4:97–105.

  15. 15.

    Darnell JE Jr., Kerr IM, Stark GR. Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science. 1994;264:1415–21.

  16. 16.

    Fu XY. From PTK-STAT signaling to caspase expression and apoptosis induction. Cell Death Differ. 1999;6:1201–8.

  17. 17.

    Guo C, Chang CC, Wortham M, Chen LH, Kernagis DN, Qin X, et al. Global identification of MLL2-targeted loci reveals MLL2’s role in diverse signaling pathways. Proc Natl Acad Sci USA. 2012;109:17603–8.

  18. 18.

    Lo H-W, Hsu S-C, Ali-Seyed M, Gunduz M, Xia W, Wei Y, et al. Nuclear interaction of EGFR and STAT3 in the activation of iNOS/NO pathway. Cancer Cell. 2005;7:575–89.

  19. 19.

    Lo HW, Hsu SC, Xia W, Cao X, Shih JY, Wei Y, et al. Epidermal growth factor receptor cooperates with signal transducer and activator of transcription 3 to induce epithelial–mesenchymal transition in cancer cells via up-regulation of TWIST gene expression. Cancer Res. 2007;67:9066–76.

  20. 20.

    Lo HW, Cao X, Zhu H, Ali-Osman F. Constitutively activated STAT3 frequently coexpresses with epidermal growth factor receptor in high-grade gliomas and targeting STAT3 sensitizes them to Iressa and alkylators. Clin Cancer Res. 2008;14:6042–54.

  21. 21.

    Lo HW, Cao X, Zhu H, Ali-Osman F. Cyclooxygenase-2 is a novel transcriptional target of the nuclear EGFR-STAT3 and EGFRvIII-STAT3 signaling axes. Mol Cancer Res. 2010;8:232–45.

  22. 22.

    Lo HW, Hsu SC, Ali-Seyed M, Gunduz M, Xia W, Wei Y, et al. Nuclear interaction of EGFR and STAT3 in the activation of the iNOS/NO pathway. Cancer Cell. 2005;7:575–89.

  23. 23.

    Clement V, Sanchez P, de Tribolet N, Radovanovic I, Ruiz i Altaba A. HEDGEHOG-GLI1 signaling regulates human glioma growth, cancer stem cell self-renewal, and tumorigenicity. Curr Biol. 2007;17:165–72.

  24. 24.

    Dahmane N, Sanchez P, Gitton Y, Palma V, Sun T, Beyna M, et al. The sonic Hedgehog-Gli pathway regulates dorsal brain growth and tumorigenesis. Development. 2001;128:5201–12.

  25. 25.

    Fiaschi M, Rozell B, Bergstrom A, Toftgard R. Development of mammary tumors by conditional expression of GLI1. Cancer Res. 2009;69:4810–7.

  26. 26.

    Kinzler KW, Ruppert JM, Bigner SH, Vogelstein B. The GLI gene is a member of the Kruppel family of zinc finger proteins. Nature. 1988;332:371–4.

  27. 27.

    Kinzler KW, Vogelstein B. The GLI gene encodes a nuclear protein which binds specific sequences in the human genome. Mol Cell Biol. 1990;10:634–42.

  28. 28.

    Rao G, Pedone CA, Del Valle L, Reiss K, Holland EC, Fults DW. Sonic hedgehog and insulin-like growth factor signaling synergize to induce medulloblastoma formation from nestin-expressing neural progenitors in mice. Oncogene. 2004;23:6156–62.

  29. 29.

    Liu S, Dontu G, Mantle ID, Patel S, Ahn NS, Jackson KW, et al. Hedgehog signaling and Bmi-1 regulate self-renewal of normal and malignant human mammary stem cells. Cancer Res. 2006;66:6063–71.

  30. 30.

    Zhu H, Lo H-W. The Human glioma-associated oncogene homolog 1 (GLI1) family of transcription factors in gene regulation and diseases. Curr Genom. 2010;11:238–45.

  31. 31.

    Kinzler KW, Bigner SH, Bigner DD, Trent JM, Law ML, O’Brien SJ, et al. Identification of an amplified, highly expressed gene in a human glioma. Science (New York, NY). 1987;236:70–73.

  32. 32.

    ten Haaf A, Bektas N, von Serenyi S, Losen I, Arweiler EC, Hartmann A, et al. Expression of the glioma-associated oncogene homolog (GLI) 1 in human breast cancer is associated with unfavourable overall survival. BMC Cancer. 2009;9:298.

  33. 33.

    Xu L, Kwon YJ, Frolova N, Steg AD, Yuan K, Johnson MR, et al. Gli1 promotes cell survival and is predictive of a poor outcome in ERalpha-negative breast cancer. Breast Cancer Res Treat. 2009;123:59–71.

  34. 34.

    Lo HW, Zhu H, Cao X, Aldrich A, Ali-Osman F. A novel splice variant of GLI1 that promotes glioblastoma cell migration and invasion. Cancer Res. 2009;69:6790–8.

  35. 35.

    Cao X, Geradts J, Dewhirst MW, Lo HW. Upregulation of VEGF-A and CD24 gene expression by the tGLI1 transcription factor contributes to the aggressive behavior of breast cancer cells. Oncogene. 2012;31:104–15.

  36. 36.

    Carpenter RL, Paw I, Zhu H, Sirkisoon S, Xing F, Watabe K, et al. The gain-of-function GLI1 transcription factor TGLI1 enhances expression of VEGF-C and TEM7 to promote glioblastoma angiogenesis. Oncotarget. 2015;6:22653–65.

  37. 37.

    Han W, Carpenter RL, Lo H-W. TGLI1 upregulates expression of VEGFR2 and VEGF-A, leading to a robust VEGF-VEGFR2 autocrine loop and cancer cell growth. Cancer Hallm. 2013;1:28–37.

  38. 38.

    Zhu H, Carpenter RL, Han W, Lo HW. The GLI1 splice variant TGLI1 promotes glioblastoma angiogenesis and growth. Cancer Lett. 2014;343:51–61.

  39. 39.

    Kameda C, Tanaka H, Yamasaki A, Nakamura M, Koga K, Sato N, et al. The Hedgehog pathway is a possible therapeutic target for patients with estrogen receptor-negative breast cancer. Anticancer Res. 2009;29:871–9.

  40. 40.

    Mukherjee S, Frolova N, Sadlonova A, Novak Z, Steg A, Page GP, et al. Hedgehog signaling and response to cyclopamine differ in epithelial and stromal cells in benign breast and breast cancer. Cancer Biol Ther. 2006;5:674–83.

  41. 41.

    Yang Q, Shen SS, Zhou S, Ni J, Chen D, Wang G, et al. STAT3 activation and aberrant ligand-dependent sonic hedgehog signaling in human pulmonary adenocarcinoma. Exp Mol Pathol. 2012;93:227–36.

  42. 42.

    Gatza ML, Lucas JE, Barry WT, Kim JW, Wang Q, Crawford MD, et al. A pathway-based classification of human breast cancer. Proc Natl Acad Sci USA. 2010;107:6994–9.

  43. 43.

    Shi T, Mazumdar T, Devecchio J, Duan ZH, Agyeman A, Aziz M, et al. cDNA microarray gene expression profiling of hedgehog signaling pathway inhibition in human colon cancer cells. PLoS ONE. 2010;5:e13054.

  44. 44.

    Peterson KA, Nishi Y, Ma W, Vedenko A, Shokri L, Zhang X, et al. Neural-specific Sox2 input and differential Gli-binding affinity provide context and positional information in Shh-directed neural patterning. Genes Dev. 2012;26:2802–16.

  45. 45.

    Durant L, Watford WT, Ramos HL, Laurence A, Vahedi G, Wei L, et al. Diverse targets of the transcription factor STAT3 contribute to T cell pathogenicity and homeostasis. Immunity. 2010;32:605–15.

  46. 46.

    Yang XP, Ghoreschi K, Steward-Tharp SM, Rodriguez-Canales J, Zhu J, Grainger JR, et al. Opposing regulation of the locus encoding IL-17 through direct, reciprocal actions of STAT3 and STAT5. Nat Immunol. 2011;12:247–54.

  47. 47.

    Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012;9:357–9.

  48. 48.

    Zhang Y, Liu T, Meyer CA, Eeckhoute J, Johnson DS, Bernstein BE, et al. Model-based analysis of ChIP-Seq (MACS). Genome Biol. 2008;9:R137.

  49. 49.

    Han W, Carpenter RL, Cao X, Lo HW. STAT1 gene expression is enhanced by nuclear EGFR and HER2 via cooperation with STAT3. Mol Carcinog. 2013;52:959–69.

  50. 50.

    Ahn S, Joyner AL. In vivo analysis of quiescent adult neural stem cells responding to Sonic hedgehog. Nature. 2005;437:894–7.

  51. 51.

    Carpenter RL, Lo HW. STAT3 target genes relevant to human. Cancers (Basel). 2014;6:897–925.

  52. 52.

    Milla LA, Gonzalez-Ramirez CN, Palma V. Sonic Hedgehog in cancer stem cells: a novel link with autophagy. Biol Res. 2012;45:223–30.

  53. 53.

    Larive RM, Moriggi G, Menacho-Marquez M, Canamero M, de Alava E, Alarcon B, et al. Contribution of the R-Ras2 GTP-binding protein to primary breast tumorigenesis and late-stage metastatic disease. Nat Commun. 2014;5:3881.

  54. 54.

    Graham SM, Oldham SM, Martin CB, Drugan JK, Zohn IE, Campbell S, et al. TC21 and Ras share indistinguishable transforming and differentiating activities. Oncogene. 1999;18:2107–16.

  55. 55.

    Clark GJ, Kinch MS, Gilmer TM, Burridge K, Der CJ. Overexpression of the Ras-related TC21/R-Ras2 protein may contribute to the development of human breast cancers. Oncogene. 1996;12:169–76.

  56. 56.

    Shi X, Sun X, Liu M, Li D, Aneja R, Zhou J. CEP70 protein interacts with gamma-tubulin to localize at the centrosome and is critical for mitotic spindle assembly. J Biol Chem. 2011;286:33401–8.

  57. 57.

    Shi X, Yao Y, Wang Y, Zhang Y, Huang Q, Zhou J, et al. Cep70 regulates microtubule stability by interacting with HDAC6. FEBS Lett. 2015;589:1771–7.

  58. 58.

    Shi X, Liu M, Li D, Wang J, Aneja R, Zhou J. Cep70 contributes to angiogenesis by modulating microtubule rearrangement and stimulating cell polarization and migration. Cell Cycle (Georgetown, TX). 2012;11:1554–63.

  59. 59.

    Shi X, Li D, Wang Y, Liu S, Qin J, Wang J, et al. Discovery of centrosomal protein 70 as an important player in the development and progression of breast cancer. Am J Pathol. 2017;187:679–88.

  60. 60.

    Kim VN, Kataoka N, Dreyfuss G. Role of the nonsense-mediated decay factor hUpf3 in the splicing-dependent exon–exon junction complex. Science (New York, NY). 2001;293:1832–6.

  61. 61.

    Shum EY, Jones SH, Shao A, Dumdie J, Krause MD, Chan WK, et al. The antagonistic gene paralogs Upf3a and Upf3b govern nonsense-mediated RNA decay. Cell. 2016;165:382–95.

  62. 62.

    Bos PD, Zhang XH, Nadal C, Shu W, Gomis RR, Nguyen DX, et al. Genes that mediate breast cancer metastasis to the brain. Nature. 2009;459:1005–9.

  63. 63.

    Mani SA, Guo W, Liao MJ, Eaton EN, Ayyanan A, Zhou AY, et al. The epithelial–mesenchymal transition generates cells with properties of stem cells. Cell. 2008;133:704–15.

  64. 64.

    Wei D, Le X, Zheng L, Wang L, Frey JA, Gao AC, et al. Stat3 activation regulates the expression of vascular endothelial growth factor and human pancreatic cancer angiogenesis and metastasis. Oncogene. 2003;22:319–29.

  65. 65.

    Sasaki H, Hui C, Nakafuku M, Kondoh H. A binding site for Gli proteins is essential for HNF-3beta floor plate enhancer activity in transgenics and can respond to Shh in vitro. Development. 1997;124:1313–22.

  66. 66.

    Carpenter RL, Paw I, Dewhirst MW, Lo HW. Akt phosphorylates and activates HSF-1 independent of heat shock, leading to Slug overexpression and epithelial–mesenchymal transition (EMT) of HER2-overexpressing breast cancer cells. Oncogene. 2015;34:546–57.

  67. 67.

    Langlois B, Saupe F, Rupp T, Arnold C, van der Heyden M, Orend G, et al. AngioMatrix, a signature of the tumor angiogenic switch-specific matrisome, correlates with poor prognosis for glioma and colorectal cancer patients. Oncotarget. 2014;5:10529–45.

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We would like to acknowledge our colleagues Dr. Shadi Qasem for providing pathology support, and funding support for this project from NIH grants R01NS087169 (to H-WL), T32CA079448 (to SK, RLC), R01NS087169-3S1 (to H-WL and SRS), P30CA012197 (to BP; core facility), and DoD grant W81XWH-17-1-0044 (to H-WL).

Author contributions

SRS and RLC conducted most of the experiments, while TR, AA, AH, and AML provided technical assistance. GJ conducted ChIP-seq data analysis, while KW provided critical scientific input during revision. H-WL and SRS wrote the original and revised manuscripts. H-WL serves as the principal investigator of this project.

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Author notes

  1. These authors contributed equally: Sherona R. Sirkisoon and Richard L. Carpenter.


  1. Department of Cancer Biology, Winston-Salem, NC, USA

    • Sherona R. Sirkisoon
    • , Richard L. Carpenter
    • , Tadas Rimkus
    • , Ashley Anderson
    • , Alexandria Harrison
    • , Allison M. Lange
    • , Kounosuke Watabe
    •  & Hui-Wen Lo
  2. Department of Radiology, Winston-Salem, NC, USA

    • Guangxu Jin
  3. Wake Forest Comprehensive Cancer Center, Wake Forest University School of Medicine, Winston-Salem, NC, USA

    • Kounosuke Watabe
    •  & Hui-Wen Lo


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The authors declare that they have no competing interests.

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Correspondence to Hui-Wen Lo.

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