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VEGF-C mediates tumor growth and metastasis through promoting EMT-epithelial breast cancer cell crosstalk


It is well established that a subset of cells within primary breast cancers can undergo an epithelial-to-mesenchymal transition (EMT), although the role of EMT in metastasis remains controversial. We previously demonstrated that breast cancer cells that had undergone an oncogenic EMT could increase metastasis of neighboring cancer cells via non-canonical paracrine-mediated activation of GLI activity that is dependent on SIX1 expression in the EMT cancer cells. However, the mechanism by which these SIX1-expressing EMT cells activate GLI signaling remained unclear. In this study, we demonstrate a novel mechanism for activation of GLI-mediated signaling in epithelial breast tumor cells via EMT cell-induced production and secretion of VEGF-C. We show that VEGF-C, secreted by breast cancer cells that have undergone an EMT, promotes paracrine-mediated increases in proliferation, migration, and invasion of epithelial breast cancer cells, via non-canonical activation of GLI-signaling. We further show that the aggressive phenotypes, including metastasis, imparted by EMT cells on adjacent epithelial cancer cells can be disrupted by either inhibiting VEGF-C in EMT cells or by knocking down NRP2, a receptor which interacts with VEGF-C, in neighboring epithelial cancer cells. Interrogation of TCGA and GEO public datasets supports the relevance of this pathway in human breast cancer, demonstrating that VEGF-C strongly correlates with activation of Hedgehog signaling and EMT in the human disease. Our study suggests that the VEGF-C/NRP2/GLI axis is a novel and conserved paracrine means by which EMT cells enhance metastasis, and provides potential targets for therapeutic intervention in this heterogeneous disease.

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Fig. 1: VEGF-C is downstream of SIX1 in three different models of EMT.
Fig. 2: GLI signaling is activated downstream of VEGF-C.
Fig. 3: VEGF-C, secreted by EMT cells, leads to increased growth of epithelial cancer cells.
Fig. 4: EMT cell-induced VEGF-C mediates aggressive phenotypes in epithelial cancer cells.
Fig. 5: NRP2 expressed on epithelial cancer cells is necessary for VEGF-C to activate GLI signaling.
Fig. 6: Vegf-c KD in Met1 (EMT) cells or Nrp2 KD in epithelial DB7 mammary carcinoma cells can inhibit Met1 induced growth and metastasis of DB7 cells in vivo.
Fig. 7: VEGFC positively correlates with Hh pathway genes in human breast tumors.
Fig. 8: EMT-epithelial cell crosstalk enhances tumor progression.


  1. 1.

    Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018;68:394–424.

    Google Scholar 

  2. 2.

    Valastyan S, Weinberg RA. Tumor metastasis: molecular insights and evolving paradigms. Cell. 2011;147:275–92.

    CAS  PubMed  PubMed Central  Google Scholar 

  3. 3.

    Aparicio S, Caldas C. The implications of clonal genome evolution for cancer medicine. N. Engl J Med. 2013;368:842–51.

    CAS  PubMed  Google Scholar 

  4. 4.

    Gerlinger M, Rowan AJ, Horswell S, Math M, Larkin J, Endesfelder D, et al. Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. N Engl J Med. 2012;366:883–92.

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5.

    Lindstrom LS, Yau C, Czene K, Thompson CK, Hoadley KA, Van’t Veer LJ, et al. Intratumor heterogeneity of the estrogen receptor and the long-term risk of fatal breast cancer. J Natl Cancer Inst. 2018;110:726–33.

    PubMed  PubMed Central  Google Scholar 

  6. 6.

    Yang F, Cao L, Sun Z, Jin J, Fang H, Zhang W, et al. Evaluation of breast cancer stem cells and intratumor stemness heterogeneity in triple-negative breast cancer as prognostic factors. Int J Biol Sci. 2016;12:1568–77.

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Yang F, Wang Y, Li Q, Cao L, Sun Z, Jin J, et al. Intratumor heterogeneity predicts metastasis of triple-negative breast cancer. Carcinogenesis. 2017;38:900–9.

    CAS  PubMed  Google Scholar 

  8. 8.

    Burrell RA, McGranahan N, Bartek J, Swanton C. The causes and consequences of genetic heterogeneity in cancer evolution. Nature. 2013;501:338–45.

    CAS  PubMed  Google Scholar 

  9. 9.

    Lin L, Lin DC. Biological significance of tumor heterogeneity in esophsageal squamous cell carcinoma. Cancers. 2019;11:1156.

    PubMed Central  Google Scholar 

  10. 10.

    McGranahan N, Swanton C. Biological and therapeutic impact of intratumor heterogeneity in cancer evolution. Cancer Cell. 2015;27:15–26.

    CAS  PubMed  Google Scholar 

  11. 11.

    Ma F, Guan Y, Yi Z, Chang L, Li Q, Chen S, et al. Assessing tumor heterogeneity using ctDNA to predict and monitor therapeutic response in metastatic breast cancer. Int J Cancer. 2020;146:1359–68.

    CAS  PubMed  Google Scholar 

  12. 12.

    Calbo J, van Montfort E, Proost N, van Drunen E, Beverloo HB, Meuwissen R, et al. A functional role for tumor cell heterogeneity in a mouse model of small cell lung cancer. Cancer Cell. 2011;19:244–56.

    CAS  PubMed  Google Scholar 

  13. 13.

    Marusyk A, Tabassum DP, Altrock PM, Almendro V, Michor F, Polyak K. Non-cell-autonomous driving of tumour growth supports sub-clonal heterogeneity. Nature. 2014;514:54–8.

    CAS  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Neelakantan D, Zhou H, Oliphant MUJ, Zhang X, Simon LM, Henke DM, et al. EMT cells increase breast cancer metastasis via paracrine GLI activation in neighbouring tumour cells. Nat Commun. 2017;8:15773.

    PubMed  PubMed Central  Google Scholar 

  15. 15.

    Chen JC, Chang YW, Hong CC, Yu YH, Su JL. The role of the VEGF-C/VEGFRs axis in tumor progression and therapy. Int J Mol Sci. 2012;14:88–107.

    PubMed  PubMed Central  Google Scholar 

  16. 16.

    Wang CA, Harrell JC, Iwanaga R, Jedlicka P, Ford HL. Vascular endothelial growth factor C promotes breast cancer progression via a novel antioxidant mechanism that involves regulation of superoxide dismutase 3. Breast Cancer Res. 2014;16:462.

    PubMed  PubMed Central  Google Scholar 

  17. 17.

    Borowsky AD, Namba R, Young LJ, Hunter KW, Hodgson JG, Tepper CG, et al. Syngeneic mouse mammary carcinoma cell lines: two closely related cell lines with divergent metastatic behavior. Clin Exp Metastasis. 2005;22:47–59.

    CAS  PubMed  Google Scholar 

  18. 18.

    Micalizzi DS, Wang CA, Farabaugh SM, Schiemann WP, Ford HL. Homeoprotein Six1 increases TGF-beta type I receptor and converts TGF-beta signaling from suppressive to supportive for tumor growth. Cancer Res. 2010;70:10371–80.

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Blevins MA, Towers CG, Patrick AN, Zhao R, Ford HL. The SIX1-EYA transcriptional complex as a therapeutic target in cancer. Expert Opin Ther Targets. 2015;19:213–25.

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Kong D, Liu Y, Liu Q, Han N, Zhang C, Pestell RG, et al. The retinal determination gene network: from developmental regulator to cancer therapeutic target. Oncotarget. 2016;7:50755–65.

    PubMed  PubMed Central  Google Scholar 

  21. 21.

    Varjosalo M, Taipale J. Hedgehog: functions and mechanisms. Genes Dev. 2008;22:2454–72.

    CAS  PubMed  Google Scholar 

  22. 22.

    Pietrobono S, Gagliardi S, Stecca B. Non-canonical Hedgehog signaling pathway in cancer: activation of GLI transcription factors beyond smoothened. Front Genet. 2019;10:556.

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Po A, Silvano M, Miele E, Capalbo C, Eramo A, Salvati V, et al. Noncanonical GLI1 signaling promotes stemness features and in vivo growth in lung adenocarcinoma. Oncogene. 2017;36:4641–52.

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Wang CA, Jedlicka P, Patrick AN, Micalizzi DS, Lemmer KC, Deitsch E, et al. SIX1 induces lymphangiogenesis and metastasis via upregulation of VEGF-C in mouse models of breast cancer. J Clin Investig. 2012;122:1895–906.

    CAS  PubMed  Google Scholar 

  25. 25.

    Liu D, Li L, Zhang XX, Wan DY, Xi BX, Hu Z, et al. SIX1 promotes tumor lymphangiogenesis by coordinating TGFbeta signals that increase expression of VEGF-C. Cancer Res. 2014;74:5597–607.

    CAS  PubMed  Google Scholar 

  26. 26.

    Goel HL, Pursell B, Chang C, Shaw LM, Mao J, Simin K, et al. GLI1 regulates a novel neuropilin-2/alpha6beta1 integrin based autocrine pathway that contributes to breast cancer initiation. EMBO Mol Med. 2013;5:488–508.

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 27.

    Wang J, Huang Y, Zhang J, Xing B, Xuan W, Wang H, et al. NRP-2 in tumor lymphangiogenesis and lymphatic metastasis. Cancer Lett. 2018;418:176–84.

    CAS  PubMed  Google Scholar 

  28. 28.

    Xu Y, Yuan L, Mak J, Pardanaud L, Caunt M, Kasman I, et al. Neuropilin-2 mediates VEGF-C-induced lymphatic sprouting together with VEGFR3. J Cell Biol. 2010;188:115–30.

    CAS  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Makinen T, Veikkola T, Mustjoki S, Karpanen T, Catimel B, Nice EC, et al. Isolated lymphatic endothelial cells transduce growth, survival and migratory signals via the VEGF-C/D receptor VEGFR-3. EMBO J. 2001;20:4762–73.

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Kalluri R, Weinberg RA. The basics of epithelial-mesenchymal transition. J Clin Investig. 2009;119:1420–8.

    CAS  PubMed  Google Scholar 

  31. 31.

    Drasin DJ, Robin TP, Ford HL. Breast cancer epithelial-to-mesenchymal transition: examining the functional consequences of plasticity. Breast Cancer Res. 2011;13:226.

    PubMed  PubMed Central  Google Scholar 

  32. 32.

    Fischer KR, Durrans A, Lee S, Sheng J, Li F, Wong ST, et al. Epithelial-to-mesenchymal transition is not required for lung metastasis but contributes to chemoresistance. Nature. 2015;527:472–6.

    CAS  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Zheng X, Carstens JL, Kim J, Scheible M, Kaye J, Sugimoto H, et al. Epithelial-to-mesenchymal transition is dispensable for metastasis but induces chemoresistance in pancreatic cancer. Nature. 2015;527:525–30.

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Micalizzi DS, Christensen KL, Jedlicka P, Coletta RD, Baron AE, Harrell JC, et al. The Six1 homeoprotein induces human mammary carcinoma cells to undergo epithelial-mesenchymal transition and metastasis in mice through increasing TGF-beta signaling. J Clin Investig. 2009;119:2678–90.

    CAS  PubMed  Google Scholar 

  35. 35.

    Liu Q, Li A, Tian Y, Liu Y, Li T, Zhang C, et al. The expression profile and clinic significance of the SIX family in non-small cell lung cancer. J Hematol Oncol. 2016;9:119.

    PubMed  PubMed Central  Google Scholar 

  36. 36.

    Towers CG, Guarnieri AL, Micalizzi DS, Harrell JC, Gillen AE, Kim J, et al. The Six1 oncoprotein downregulates p53 via concomitant regulation of RPL26 and microRNA-27a-3p. Nat Commun. 2015;6:10077.

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Ford HL, Kabingu EN, Bump EA, Mutter GL, Pardee AB. Abrogation of the G2 cell cycle checkpoint associated with overexpression of HSIX1: a possible mechanism of breast carcinogenesis. Proc Natl Acad Sci USA. 1998;95:12608–13.

    CAS  PubMed  Google Scholar 

  38. 38.

    Joukov V, Pajusola K, Kaipainen A, Chilov D, Lahtinen I, Kukk E, et al. A novel vascular endothelial growth factor, VEGF-C, is a ligand for the Flt4 (VEGFR-3) and KDR (VEGFR-2) receptor tyrosine kinases. EMBO J. 1996;15:1751.

    CAS  PubMed  PubMed Central  Google Scholar 

  39. 39.

    Cao Y, Linden P, Farnebo J, Cao R, Eriksson A, Kumar V, et al. Vascular endothelial growth factor C induces angiogenesis in vivo. Proc Natl Acad Sci USA. 1998;95:14389–94.

    CAS  PubMed  Google Scholar 

  40. 40.

    Skobe M, Hawighorst T, Jackson DG, Prevo R, Janes L, Velasco P, et al. Induction of tumor lymphangiogenesis by VEGF-C promotes breast cancer metastasis. Nat Med. 2001;7:192–8.

    CAS  PubMed  Google Scholar 

  41. 41.

    Lund AW, Duraes FV, Hirosue S, Raghavan VR, Nembrini C, Thomas SN, et al. VEGF-C promotes immune tolerance in B16 melanomas and cross-presentation of tumor antigen by lymph node lymphatics. Cell Rep. 2012;1:191–9.

    CAS  PubMed  Google Scholar 

  42. 42.

    Yeh YW, Cheng CC, Yang ST, Tseng CF, Chang TY, Tsai SY, et al. Targeting the VEGF-C/VEGFR3 axis suppresses Slug-mediated cancer metastasis and stemness via inhibition of KRAS/YAP1 signaling. Oncotarget. 2017;8:5603–18.

    PubMed  Google Scholar 

  43. 43.

    Dias S, Choy M, Alitalo K, Rafii S. Vascular endothelial growth factor (VEGF)-C signaling through FLT-4 (VEGFR-3) mediates leukemic cell proliferation, survival, and resistance to chemotherapy. Blood. 2002;99:2179–84.

    CAS  PubMed  Google Scholar 

  44. 44.

    Su JL, Yen CJ, Chen PS, Chuang SE, Hong CC, Kuo IH, et al. The role of the VEGF-C/VEGFR-3 axis in cancer progression. Br J Cancer. 2007;96:541–5.

    CAS  PubMed  Google Scholar 

  45. 45.

    Kampen KR, Scherpen FJG, Mahmud H, Ter Elst A, Mulder AB, Guryev V, et al. VEGFC antibody therapy drives differentiation of AML. Cancer Res. 2018;78:5940–8.

    CAS  PubMed  Google Scholar 

  46. 46.

    Lin J, Lalani AS, Harding TC, Gonzalez M, Wu WW, Luan B, et al. Inhibition of lymphogenous metastasis using adeno-associated virus-mediated gene transfer of a soluble VEGFR-3 decoy receptor. Cancer Res. 2005;65:6901–9.

    CAS  PubMed  Google Scholar 

  47. 47.

    Zhang D, Li B, Shi J, Zhao L, Zhang X, Wang C, et al. Suppression of tumor growth and metastasis by simultaneously blocking vascular endothelial growth factor (VEGF)-A and VEGF-C with a receptor-immunoglobulin fusion protein. Cancer Res. 2010;70:2495–503.

    CAS  PubMed  Google Scholar 

  48. 48.

    Wang CA, Tsai SJ. The non-canonical role of vascular endothelial growth factor-C axis in cancer progression. Exp Biol Med. 2015;240:718–24.

    CAS  Google Scholar 

  49. 49.

    Pinskey JM, Franks NE, McMellen AN, Giger RJ, Allen BL. Neuropilin-1 promotes Hedgehog signaling through a novel cytoplasmic motif. J Biol Chem. 2017;292:15192–204.

    CAS  PubMed  PubMed Central  Google Scholar 

  50. 50.

    Apte RS, Chen DS, Ferrara N. VEGF in signaling and disease: beyond discovery and development. Cell. 2019;176:1248–64.

    CAS  PubMed  PubMed Central  Google Scholar 

  51. 51.

    Jimeno A, Weiss GJ, Miller WH Jr., Gettinger S, Eigl BJ, Chang AL, et al. Phase I study of the Hedgehog pathway inhibitor IPI-926 in adult patients with solid tumors. Clin Cancer Res. 2013;19:2766–74.

    CAS  PubMed  PubMed Central  Google Scholar 

  52. 52.

    Takebe N, Miele L, Harris PJ, Jeong W, Bando H, Kahn M, et al. Targeting Notch, Hedgehog, and Wnt pathways in cancer stem cells: clinical update. Nat Rev Clin Oncol. 2015;12:445–64.

    CAS  PubMed  PubMed Central  Google Scholar 

  53. 53.

    Amakye D, Jagani Z, Dorsch M. Unraveling the therapeutic potential of the Hedgehog pathway in cancer. Nat Med. 2013;19:1410–22.

    CAS  PubMed  Google Scholar 

  54. 54.

    Wei T, Simko V. R package “corrplot”: visualization of a correlation matrix (Version 0.84), 2017.

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This work was supported by R01CA224867 to HLF and MTL, and F99CA234940 to HZ. We would like to thank the Functional Genomics Shared Resource (SR), the Cell Technologies SR, and the Animal Imaging SR of the University of Colorado Cancer Center (P30CA046934) for help with these studies. We would also like to acknowledge support from The Dan L. Duncan Comprehensive Cancer Center (BCM, P30CA125123) and the Cancer Prevention and Research Initiative of Texas (CPRIT) (BCM, RP170691).

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Correspondence to Heide L. Ford.

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MTL is a Founder and Limited Partner in StemMed Ltd, and a Founder and Manager in StemMed Holdings, its General Partner. He is also a Founder and equity stake holder in Tvardi Therapeutics Inc. The remaining authors do not have any conflicts of interest.

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Kong, D., Zhou, H., Neelakantan, D. et al. VEGF-C mediates tumor growth and metastasis through promoting EMT-epithelial breast cancer cell crosstalk. Oncogene 40, 964–979 (2021).

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