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Phosphorylation of EphA2 receptor and vasculogenic mimicry is an indicator of poor prognosis in invasive carcinoma of the breast

  • Debarpan Mitra
  • Sayantan Bhattacharyya
  • Neyaz Alam
  • Sagar Sen
  • Saunak Mitra
  • Syamsundar Mandal
  • Shivani Vignesh
  • Biswanath Majumder
  • Nabendu MurmuEmail author
Preclinical study
  • 39 Downloads

Abstract

Purpose

The occurrence of vasculogenic mimicry (VM) and EphA2-mediated tumour progression are associated with poor prognosis in various solid tumours. Here, we aimed to investigate the prognostic implications of VM and its association with phosphorylated EphA2 receptor in invasive carcinoma of the breast.

Methods

The patients were stratified based on CD-31/PAS dual staining and subsequently the expression status of phospho-EphA2 (S897), FAK, phospho-ERK1/2 and Laminin 5Ƴ2 was analysed by immunohistochemistry. Survival of patients was correlated within the stratified cohort.

Results

The pathologically defined VM phenotype and phospho-EphA2 (S897) expression status were significantly associated with lower disease-free survival (DFS) and overall survival (OS). Both the features were also found to be significantly associated with higher nodal status, poor Nottingham Prognostic Index (NPI) and were more prevalent in the triple-negative breast cancer (TNBC) group. Incidentally, there were no significant association between age of the patient, grade and size of the tumour with VM and phospho-EphA2 (S897). The effector molecules of phospho-EphA2 (S897) viz., Focal Adhesion Kinase (FAK), phospho-ERK1/2 and Laminin 5Ƴ2 were significantly upregulated in the VM-positive cohort. Survival analysis revealed that the VM and phospho-EphA2 (S897) dual-positive cohort had poorest DFS [mean time = 48.313 (39.992–56.633) months] and OS [mean time = 56.692 (49.055–64.328) months]. Individually, VM-positive [Hazard Ratio (HR) 6.005; 95% confidence interval (CI) 2.002–18.018; P = 0.001 for DFS and HR 11.654; 95% CI 3.195–42.508; P < 0.0001 for OS] and phospho-EphA2 (S897)-positive (HR 4.342; 95% CI 1.717–10.983; P = 0.002 for DFS and HR 5.853; 95% CI 1.663–20.602; P = 0.006 for OS) expression proved to be independent indicators of prognosis.

Conclusion

This study evaluated tumour dependency on oncogenic EphA2 receptor regulation and VM in invasive carcinoma of the breast and their prognostic significance. Significant correlations between VM, phospho-EphA2 and several clinicopathologic parameters of breast cancer were found. Subsequently, the occurrence of VM or phospho-EphA2 expression proved to be major contributors for poor prognosis in patients with breast cancer but their simultaneous expression failed to be an independent risk factor.

Keywords

Breast cancer Vasculogenic mimicry EphA2 Prognosis Overall survival 

Notes

Acknowledgements

We wish to thank Dr. Jayanta Chakrabarti, Director, CNCI, for his immense support throughout the project.

Author contributions

DM and NM conceptualised and designed the study. DM and SB performed IHC experiments and collected patient data. DM and SSM analysed the data. DM, SM, SV, BM and NM interpreted the data. NA and SS provided clinical insights and interpreted clinical data and did follow-up of patients. DM, SB, BM and NM wrote the manuscript.

Funding

This project was supported by Chittaranjan National Cancer Institute, Kolkata (Intramural fund, Sanction No. A-4.482/2017/271).

Compliance with ethical standards

Conflict of interest

Authors have no conflict of interest to declare.

Ethical approval

This study was approved by the Institutional ethics committee at Chittaranjan National Cancer Institute, Kolkata (IEC Ref: A-4.311/NM/26/11/2018). All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.

Informed consent

Written informed consent was not required as this was a retrospective study and patient information was de-identified prior to analysis.

Supplementary material

10549_2019_5482_MOESM1_ESM.tif (888 kb)
Supplementary material 1 (TIFF 888 kb)
10549_2019_5482_MOESM2_ESM.doc (33 kb)
Supplementary material 2 (DOC 33 kb)
10549_2019_5482_MOESM3_ESM.doc (32 kb)
Supplementary material 3 (DOC 32 kb)

References

  1. 1.
    Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A (2018) Global Cancer Statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 Cancers in 185 countries. Cancer J Clin 68:394–424CrossRefGoogle Scholar
  2. 2.
    Yam C, Mani SA, Moulder SL (2017) Targeting the molecular subtypes of triple negative breast cancer: understanding the diversity to progress the field. Oncologist 22(9):1086–1093PubMedPubMedCentralCrossRefGoogle Scholar
  3. 3.
    Janic B, Arbab AS (2010) The role and therapeutic potential of endothelial progenitor cells in tumor neovascularization. Sci World J 10:1088–1099CrossRefGoogle Scholar
  4. 4.
    Servick K (2014) Breast cancer. Breast cancer: a world of differences. Science 343:1452PubMedCrossRefGoogle Scholar
  5. 5.
    Kumar N, Prasad P, Jash E, Jayasundar S, Singh I, Alam N, Murmu N, Somashekhar SP, Goldman A, Sehrawat S (2018) cAMP regulated EPAC1 supports microvascular density, angiogenic and metastatic properties in a model of triple negative breast cancer. Carcinogenesis 39:1245–1253PubMedPubMedCentralCrossRefGoogle Scholar
  6. 6.
    Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144:646–674CrossRefGoogle Scholar
  7. 7.
    Yousef Ahmed Fouad and Carmen Aanei (2017) Revisiting the hallmarks of cancer. Am J Cancer Res 7(5):1016–1036PubMedGoogle Scholar
  8. 8.
    Robert NJ, Dieras V, Glaspy J, Brufsky AM, Bondarenko I, Lipatov ON, Perez EA, Yardley DA, Chan SY, Zhou X, Phan SC, O’Shaughnessy J (2011) RIBBON-1: randomized, double-blind, placebo-controlled, phase III trial of chemotherapy with or without bevacizumab for first-line treatment of human epidermal growth factor receptor 2-negative, locally recurrent or metastatic breast cancer. J Clin Oncol 29:1252–1260PubMedCrossRefGoogle Scholar
  9. 9.
    Rochlitz C, Bigler M, von Moos R, Bernhard J, Matter- Walstra K, Wicki A, Zaman K, Anchisi S, Kung M, Na KJ, Bartschi D, Borner M, Rordorf T et al (2016) SAKK 24/09: safety and tolerability of bevacizumab plus paclitaxel vs. bevacizumab plus metronomic cyclophosphamide and capecitabine as first-line therapy in patients with HER2- negative advanced stage breast cancer—a multicenter, randomized phase III trial. BMC Cancer 16:780PubMedPubMedCentralCrossRefGoogle Scholar
  10. 10.
    Dickler MN, Barry WT, Cirrincione CT, Ellis MJ, Moynahan ME, Innocenti F, Hurria A, Rugo HS, Lake DE, Hahn O, Schneider BP, Tripathy D, Carey LA et al (2016) Phase III trial evaluating letrozole as first-line endocrine therapy with or without bevacizumab for the treatment of postmenopausal women with hormone receptor-positive advanced-stage breast cancer: CALGB 40503 (alliance). J Clin Oncol 34:2602–2609PubMedPubMedCentralCrossRefGoogle Scholar
  11. 11.
    Maniotis AJ, Folberg R, Hess A, Seftor EA, Gardner LM, Peer J, Trent JM, Meltzer PS, Hendrix MJ (1999) Vascular channel formation by human melanoma cells in vivo and in vitro: vasculogenic mimicry. Am J Pathol. 155:739–752PubMedPubMedCentralCrossRefGoogle Scholar
  12. 12.
    Ge Hong, Luo Hui (2018) Overview of advances in vasculogenic mimicry—a potential target for tumor therapy. Cancer Manag Res. 10:2429–2437PubMedPubMedCentralCrossRefGoogle Scholar
  13. 13.
    Qiao L, Liang N, Zhang J, Xie J, Liu F, Xu D, Yu X, Tian Y (2015) Advanced research on vasculogenic mimicry in cancer. J Cell Mol Med 19:315–326PubMedPubMedCentralCrossRefGoogle Scholar
  14. 14.
    Bhattacharyya S, Mitra D, Ray S, Biswas N, Banerjee S, Majumder B, Mustafi SM, Murmu N (2019) Reversing effect of Lupeol on vasculogenic mimicry in murine melanoma progression. Microvasc Res 121:52–62PubMedCrossRefPubMedCentralGoogle Scholar
  15. 15.
    Wu S, Yu L, Cheng Z, Song W, Zhou L, Tao Y (2012) Expression of maspin in non-small cell lung cancer and its relationship to vasculogenic mimicry. J Huazhong Univ Sci Technol Med Sci. 32:346–352PubMedCrossRefPubMedCentralGoogle Scholar
  16. 16.
    Liu WB, Xu GL, Jia WD, Li JS, Ma JL, Chen K, Wang ZH, Ge YS, Ren WH, Yu JH, Wang W, Wang XJ (2011) Prognostic significance and mechanisms of patterned matrix vasculogenic mimicry in hepatocellular carcinoma. Med Oncol 28:S228–S238PubMedCrossRefPubMedCentralGoogle Scholar
  17. 17.
    Li M, Gu Y, Zhang Z, Zhang S, Zhang D, Saleem AF, Zhao X, Sun B (2010) Vasculogenic mimicry: a new prognostic sign of gastric adenocarcinoma. Pathol Oncol Res 16:259–266PubMedCrossRefPubMedCentralGoogle Scholar
  18. 18.
    Sun Q, Zou X, Zhang T, Shen J, Yin Y, Xiang J (2014) The role of miR-200a in vasculogenic mimicry and its clinical significance in ovarian cancer. Gynecol Oncol 132:730–738PubMedCrossRefPubMedCentralGoogle Scholar
  19. 19.
    Liu R, Yang K, Meng C, Zhang Z, Xu Y (2012) Vasculogenic mimicry is a marker of poor prognosis in prostate cancer. Cancer Biol Ther 13:527–533PubMedCrossRefPubMedCentralGoogle Scholar
  20. 20.
    Gong W, Sun B, Zhao X, Zhang D, Sun J, Liu T, Gu Q, Dong X, Liu F, Wang Y, Lin X, Li Y (2016) Nodal signalling promotes vasculogenic mimicry formation in breast cancer via the Smad2/3 pathway. Oncotarget 7:70152–70167PubMedPubMedCentralGoogle Scholar
  21. 21.
    Liu Y, Sun B, Liu T, Zhao X, Wang X, Li Y, Meng J, Gu Q, Liu F, Dong X, Liu P, Sun R, Zhao N (2016) Function of AURKA protein kinase in the formation of vasculogenic mimicry in triple-negative breast cancer stem cells. Onco Targets Ther 13(9):3473–3484Google Scholar
  22. 22.
    Boyd AW, Bartlett PF, Lackmann M (2014) Therapeutic targeting of EPH receptors and their ligands. Nat Rev Drug Discov 13(1):39–62PubMedCrossRefPubMedCentralGoogle Scholar
  23. 23.
    Miao H et al (2009) EphA2 mediates ligand-dependent inhibition and ligand independent promotion of cell migration and invasion via a reciprocal regulatory loop with Akt. Cancer Cell 16:9–20PubMedPubMedCentralCrossRefGoogle Scholar
  24. 24.
    Barquilla A, Pasquale EB (2015) Eph receptors and ephrins: therapeutic opportunities. Annu Rev Pharmacol Toxicol 55:465–487PubMedCrossRefPubMedCentralGoogle Scholar
  25. 25.
    Pasquale EB (2010) Eph receptors and ephrins in cancer: bidirectional signalling and beyond. Nat Rev Cancer 10:165–180PubMedPubMedCentralCrossRefGoogle Scholar
  26. 26.
    Pasquale EB (2008) Eph-ephrin bidirectional signaling in physiology and disease. Cell 133:38–52PubMedCrossRefPubMedCentralGoogle Scholar
  27. 27.
    Ireton RC, Chen J (2005) EphA2 receptor tyrosine kinase as a promising target for cancer therapeutics. Curr Cancer Drug Targets 5:149–157PubMedCrossRefPubMedCentralGoogle Scholar
  28. 28.
    Barquilla A et al (2016) Protein kinase A can block EphA2 receptor-mediated cell repulsion by increasing EphA2 S897 phosphorylation. Mol Biol Cell 27:2757–2770PubMedPubMedCentralCrossRefGoogle Scholar
  29. 29.
    Zhou Y et al (2015) Crucial roles of RSK in cell motility by catalysing serine phosphorylation of EphA2. Nat Commun 6:7679PubMedPubMedCentralCrossRefGoogle Scholar
  30. 30.
    Brantley-Sieders DM, Jiang A, Sarma K, Badu-Nkansah A, Walter DL, Shyr Y et al (2011) Eph/ephrin profiling in human breast cancer reveals significant associations between expression level and clinical outcome. PLoS One 6:e24426PubMedPubMedCentralCrossRefGoogle Scholar
  31. 31.
    Hess AR, Seftor EA, Gruman LM, Kinch MS, Seftor RE, Hendrix MJ (2006) VE-cadherin regulates EphA2 in aggressive melanoma cells through a novel signaling pathway: implications for vasculogenic mimicry. Cancer Biol Ther 5(2):228–233PubMedCrossRefGoogle Scholar
  32. 32.
    Wang H, Lin H, Pan J, Mo C, Zhang F, Huang B, Wang Z, Chen X, Zhuang J, Wang D, Qiu S (2016) Vasculogenic mimicry in prostate cancer: the roles of EphA2 and PI3K. J Cancer. 7(9):1114–1124PubMedPubMedCentralCrossRefGoogle Scholar
  33. 33.
    Wang W, Lin P, Sun B, Zhang S, Cai W, Han C, Li L, Lu H, Zhao X (2014) Epithelial-mesenchymal transition regulated by EphA2 contributes to vasculogenic mimicry formation of head and neck squamous cell carcinoma. Biomed Res Int 2014:803914PubMedPubMedCentralGoogle Scholar
  34. 34.
    Kim HS, Won YJ, Shim JH, Kim HJ, Kim J, Hong HN, Kim BS (2019) Morphological characteristics of vasculogenic mimicry and its correlation with EphA2 expression in gastric adenocarcinoma. Sci Rep 9(1):3414PubMedPubMedCentralCrossRefGoogle Scholar
  35. 35.
    Wang JY, Sun T, Zhao XL et al (2008) Functional significance of VEGF-a in human ovarian carcinoma: role in vasculogenic mimicry. Cancer Biol Ther 7:758–766PubMedCrossRefPubMedCentralGoogle Scholar
  36. 36.
    Harada K, Negishi M, Katoh H (2015) HGF-induced serine 897 phosphorylation of EphA2 regulates epithelial morphogenesis of MDCK cells in 3D culture. J Cell Sci 128:1912–1921PubMedCrossRefGoogle Scholar
  37. 37.
    Delgado-Bellido D, Serrano-Saenz S, Fernández-Cortés M, Oliver FJ (2017) Vasculogenic mimicry signaling revisited: focus on non-vascular VE-cadherin. Mol Cancer. 16(1):65PubMedPubMedCentralCrossRefGoogle Scholar
  38. 38.
    Buijs JT, Cleton AM, Smit VT, Löwik CW, Papapoulos S, Pluijm G (2004) Prognostic significance of periodic acid-Schiff-positive patterns in primary breast cancer and its lymph node metastases. Breast Cancer Res Treat 84(2):117–130PubMedCrossRefGoogle Scholar
  39. 39.
    Sun B, Zhang S, Zhang D, Li Y, Zhao X, Luo Y, Guo Y (2008) Identification of metastasis-related proteins and their clinical relevance to triple-negative human breast cancer. Clin Cancer Res 14(21):7050–7059PubMedCrossRefPubMedCentralGoogle Scholar
  40. 40.
    Sun H, Zhang D, Yao Z, Lin X, Liu J, Gu Q, Dong X, Liu F, Wang Y, Yao N, Cheng S, Li L, Sun S (2017) Anti-angiogenic treatment promotes triple-negative breast cancer invasion via vasculogenic mimicry. Cancer Biol Ther. 18(4):205–213PubMedPubMedCentralCrossRefGoogle Scholar
  41. 41.
    Kollias J, Murphy CA, Elston CW, Ellis IO, Robertson JF, Blamey RW (1999) The prognosis of small primary breast cancers. Eur J Cancer 35(6):908–912PubMedCrossRefPubMedCentralGoogle Scholar
  42. 42.
    Seftor RE, Hess AR, Seftor EA, Kirschmann DA, Hardy KM, Margaryan NV, Hendrix MJ (2012) Tumor cell vasculogenic mimicry: from controversy to therapeutic promise. Am J Pathol 181(4):1115–1125PubMedPubMedCentralCrossRefGoogle Scholar
  43. 43.
    Xing P, Dong H, Liu Q, Zhao T, Yao F, Xu Y, Chen B, Zheng X, Wu Y, Jin F, Li J (2018) ALDH1 expression and vasculogenic mimicry are positively associated with poor prognosis in patients with breast cancer. Cell Physiol Biochem 49(3):961–970PubMedCrossRefPubMedCentralGoogle Scholar
  44. 44.
    Zelinski DP, Zantek ND, Stewart JC, Irizarry AR, Kinch MS (2001) EphA2 overexpression causes tumorigenesis of mammary epithelial cells. Cancer Res 61:2301–2306PubMedPubMedCentralGoogle Scholar
  45. 45.
    Pan M (2005) Overexpression of EphA2 gene in invasive human breast cancer and its association with hormone receptor status [ASCO Annual Meeting Proceedings]. J Clin Oncol 23:9583CrossRefGoogle Scholar
  46. 46.
    Larsen AB, Pedersen MW, Stockhausen MT, Grandal MV, van Deurs B, Poulsen HS (2007) Activation of the EGFR gene target EphA2 inhibits epidermal growth factor-induced cancer cell motility. Mol Cancer Res 5:283–293PubMedCrossRefPubMedCentralGoogle Scholar
  47. 47.
    Brantley-Sieders DM, Zhuang G, Hicks D, Fang WB, Hwang Y, Cates JM, Coffman K, Jackson D, Bruckheimer E, Muraoka-Cook RS, Chen J (2008) The receptor tyrosine kinase EphA2 promotes mammary adenocarcinoma tumorigenesis and metastatic progression in mice by amplifying ErbB2 signaling. J Clin Invest 118:64–78PubMedCrossRefPubMedCentralGoogle Scholar
  48. 48.
    Vaught D, Brantley-Sieders DM, Chen J (2008) Eph receptors in breast cancer: roles in tumor promotion and tumor suppression. Breast Cancer Res 10(6):217PubMedPubMedCentralCrossRefGoogle Scholar
  49. 49.
    Miao H, Gale NW, Guo H, Qian J, Petty A, Kaspar J, Murphy AJ, Valenzuela DM, Yancopoulos G, Hambardzumyan D, Lathia JD, Rich JN, Lee J, Wang B (2015) EphA2 promotes infiltrative invasion of glioma stem cells in vivo through cross-talk with Akt and regulates stem cell properties. Oncogene 34:558–567PubMedCrossRefPubMedCentralGoogle Scholar
  50. 50.
    Huang J, Xiao D, Li G, Ma J, Chen P, Yuan W, Hou F, Ge J, Zhong M, Tang Y, Xia X, Chen Z (2014) EphA2 promotes epithelial-mesenchymal transition through the Wnt/beta-catenin pathway in gastric cancer cells. Oncogene 33:2737–2747PubMedCrossRefPubMedCentralGoogle Scholar
  51. 51.
    Binda E, Visioli A, Giani F, Lamorte G, Copetti M, Pitter KL, Huse JT, Cajola L, Zanetti N, DiMeco F, De Filippis L, Mangiola A, Maira G, Anile C, De Bonis P, Reynolds BA, Pasquale EB, Vescovi AL (2012) The EphA2 receptor drives self-renewal and tumorigenicity in stem-like tumor-propagating cells from human glioblastomas. Cancer Cell 22:765–780PubMedPubMedCentralCrossRefGoogle Scholar
  52. 52.
    Tawadros T, Brown MD, Hart CA, Clarke NW (2012) Ligand-independent activation of EphA2 by arachidonic acid induces metastasis-like behaviour in prostate cancer cells. Br J Cancer 107(10):1737–1744PubMedPubMedCentralCrossRefGoogle Scholar
  53. 53.
    Carmeliet P, Jain RK (2011) Molecular mechanisms and clinical applications of angiogenesis. Nature 473:298–307PubMedPubMedCentralCrossRefGoogle Scholar
  54. 54.
    Potente M, Gerhardt H, Carmeliet P (2011) Basic and therapeutic aspects of angiogenesis. Cell 146:873–887PubMedCrossRefPubMedCentralGoogle Scholar
  55. 55.
    Network Cancer Genome Atlas (2012) Comprehensive molecular portraits of human breast tumours. Nature 490:61–70CrossRefGoogle Scholar
  56. 56.
    Perou CM, Sorlie 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, Lonning PE, Borresen-Dale AL, Brown PO, Botstein D (2000) Molecular portraits of human breast tumours. Nature 406:747–752PubMedCrossRefPubMedCentralGoogle Scholar
  57. 57.
    Lal P, Tan LK, Chen B (2005) Correlation of HER-2 status with estrogen and progesterone receptors and histologic features in 3,655 invasive breast carcinomas. Am J Clin Pathol 123:541–546PubMedCrossRefPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Signal Transduction and Biogenic AminesChittaranjan National Cancer InstituteKolkataIndia
  2. 2.Department of Surgical OncologyChittaranjan National Cancer InstituteKolkataIndia
  3. 3.Department of PathologyChittaranjan National Cancer InstituteKolkataIndia
  4. 4.Department of Epidemiology and BiostatisticsChittaranjan National Cancer InstituteKolkataIndia
  5. 5.Department of Molecular PathologyMitra BiotechBengaluruIndia
  6. 6.Department of Cancer BiologyMitra BiotechBengaluruIndia

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