Skip to main content

Advertisement

SpringerLink
Log in

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

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

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.

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

Similar content being viewed by others

References

  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 68:394–424

    Google Scholar 

  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):1086–1093

    PubMed  PubMed Central  Google Scholar 

  3. Janic B, Arbab AS (2010) The role and therapeutic potential of endothelial progenitor cells in tumor neovascularization. Sci World J 10:1088–1099

    CAS  Google Scholar 

  4. Servick K (2014) Breast cancer. Breast cancer: a world of differences. Science 343:1452

    PubMed  Google Scholar 

  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 39:1245–1253

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144:646–674

    CAS  Google Scholar 

  7. Yousef Ahmed Fouad and Carmen Aanei (2017) Revisiting the hallmarks of cancer. Am J Cancer Res 7(5):1016–1036

    PubMed  Google Scholar 

  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 29:1252–1260

    CAS  PubMed  Google Scholar 

  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 16:780

    PubMed  PubMed Central  Google Scholar 

  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 34:2602–2609

    CAS  PubMed  PubMed Central  Google Scholar 

  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. 155:739–752

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Ge Hong, Luo Hui (2018) Overview of advances in vasculogenic mimicry—a potential target for tumor therapy. Cancer Manag Res. 10:2429–2437

    CAS  PubMed  PubMed Central  Google Scholar 

  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 19:315–326

    PubMed  PubMed Central  Google Scholar 

  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 121:52–62

    CAS  PubMed  Google Scholar 

  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. 32:346–352

    CAS  PubMed  Google Scholar 

  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–S238

    PubMed  Google Scholar 

  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 16:259–266

    PubMed  Google Scholar 

  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 132:730–738

    CAS  PubMed  Google Scholar 

  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 13:527–533

    CAS  PubMed  Google Scholar 

  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 7:70152–70167

    PubMed  PubMed Central  Google Scholar 

  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):3473–3484

    Google Scholar 

  22. Boyd AW, Bartlett PF, Lackmann M (2014) Therapeutic targeting of EPH receptors and their ligands. Nat Rev Drug Discov 13(1):39–62

    CAS  Google Scholar 

  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 16:9–20

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Barquilla A, Pasquale EB (2015) Eph receptors and ephrins: therapeutic opportunities. Annu Rev Pharmacol Toxicol 55:465–487

    CAS  PubMed  Google Scholar 

  25. Pasquale EB (2010) Eph receptors and ephrins in cancer: bidirectional signalling and beyond. Nat Rev Cancer 10:165–180

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Pasquale EB (2008) Eph-ephrin bidirectional signaling in physiology and disease. Cell 133:38–52

    CAS  PubMed  Google Scholar 

  27. Ireton RC, Chen J (2005) EphA2 receptor tyrosine kinase as a promising target for cancer therapeutics. Curr Cancer Drug Targets 5:149–157

    CAS  PubMed  Google Scholar 

  28. Barquilla A et al (2016) Protein kinase A can block EphA2 receptor-mediated cell repulsion by increasing EphA2 S897 phosphorylation. Mol Biol Cell 27:2757–2770

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Zhou Y et al (2015) Crucial roles of RSK in cell motility by catalysing serine phosphorylation of EphA2. Nat Commun 6:7679

    PubMed  PubMed Central  Google Scholar 

  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:e24426

    CAS  PubMed  PubMed Central  Google Scholar 

  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):228–233

    CAS  PubMed  Google Scholar 

  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):1114–1124

    CAS  PubMed  PubMed Central  Google Scholar 

  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 2014:803914

    PubMed  PubMed Central  Google Scholar 

  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):3414

    PubMed  PubMed Central  Google Scholar 

  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 7:758–766

    CAS  PubMed  Google Scholar 

  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 128:1912–1921

    CAS  PubMed  Google Scholar 

  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):65

    PubMed  PubMed Central  Google Scholar 

  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):117–130

    PubMed  Google Scholar 

  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):7050–7059

    CAS  PubMed  Google Scholar 

  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):205–213

    CAS  PubMed  PubMed Central  Google Scholar 

  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):908–912

    CAS  PubMed  Google Scholar 

  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):1115–1125

    CAS  PubMed  PubMed Central  Google Scholar 

  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):961–970

    CAS  PubMed  Google Scholar 

  44. Zelinski DP, Zantek ND, Stewart JC, Irizarry AR, Kinch MS (2001) EphA2 overexpression causes tumorigenesis of mammary epithelial cells. Cancer Res 61:2301–2306

    CAS  PubMed  Google Scholar 

  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 23:9583

    Google Scholar 

  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 5:283–293

    CAS  PubMed  Google Scholar 

  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 118:64–78

    CAS  PubMed  Google Scholar 

  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):217

    PubMed  PubMed Central  Google Scholar 

  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 34:558–567

    CAS  PubMed  Google Scholar 

  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 33:2737–2747

    CAS  PubMed  Google Scholar 

  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 22:765–780

    CAS  PubMed  PubMed Central  Google Scholar 

  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):1737–1744

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Carmeliet P, Jain RK (2011) Molecular mechanisms and clinical applications of angiogenesis. Nature 473:298–307

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Potente M, Gerhardt H, Carmeliet P (2011) Basic and therapeutic aspects of angiogenesis. Cell 146:873–887

    CAS  PubMed  Google Scholar 

  55. Network Cancer Genome Atlas (2012) Comprehensive molecular portraits of human breast tumours. Nature 490:61–70

    Google Scholar 

  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 406:747–752

    CAS  Google Scholar 

  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 123:541–546

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

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

Funding

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

Author information

Authors and Affiliations

  1. Department of Signal Transduction and Biogenic Amines, Chittaranjan National Cancer Institute, 37, S. P. Mukherjee Road, Kolkata, 700026, India

    Debarpan Mitra, Sayantan Bhattacharyya & Nabendu Murmu

  2. Department of Surgical Oncology, Chittaranjan National Cancer Institute, 37, S. P. Mukherjee Road, Kolkata, 700026, India

    Neyaz Alam & Sagar Sen

  3. Department of Pathology, Chittaranjan National Cancer Institute, 37, S. P. Mukherjee Road, Kolkata, 700026, India

    Saunak Mitra

  4. Department of Epidemiology and Biostatistics, Chittaranjan National Cancer Institute, 37, S. P. Mukherjee Road, Kolkata, 700026, India

    Syamsundar Mandal

  5. Department of Molecular Pathology, Mitra Biotech, 7- Service Road, Pragathi Nagar, Electronic City, Bengaluru, 560100, India

    Biswanath Majumder

  6. Department of Cancer Biology, Mitra Biotech, 7- Service Road, Pragathi Nagar, Electronic City, Bengaluru, 560100, India

    Shivani Vignesh & Biswanath Majumder

Authors
  1. Debarpan Mitra
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sayantan Bhattacharyya
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Neyaz Alam
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sagar Sen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Saunak Mitra
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Syamsundar Mandal
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Shivani Vignesh
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Biswanath Majumder
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Nabendu Murmu
    View author publications

    You can also search for this author in PubMed Google Scholar

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.

Corresponding author

Correspondence to Nabendu Murmu.

Ethics declarations

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.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Mitra, D., Bhattacharyya, S., Alam, N. et al. Phosphorylation of EphA2 receptor and vasculogenic mimicry is an indicator of poor prognosis in invasive carcinoma of the breast. Breast Cancer Res Treat 179, 359–370 (2020). https://doi.org/10.1007/s10549-019-05482-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10549-019-05482-8

Keywords

Search

Navigation