Skip to main content

Effects of MDM2 inhibitors on vascular endothelial growth factor-mediated tumor angiogenesis in human breast cancer

Abstract

Background

Mouse double minute 2 (MDM2) is overexpressed in many malignant tumors, and MDM2 levels are associated with poor prognosis of several human cancers, including breast cancer. In the present study, we investigated the function of MDM2 in vascular endothelial growth factor (VEGF)-mediated tumor angiogenesis of breast cancer and the potential value of MDM2 as an anti-angiogenic therapy target for cancer therapy by inhibiting MDM2 with antisense oligonucleotides (ASO) or other antagonist nutlin-3.

Methods

Anti-MDM2 ASO and nutlin-3 were evaluated for their in vitro and in vivo anti-angiogenesis activities in different human breast cancer models with a different p53 status: MCF-7 cell line containing wild-type p53 and MDA-MB-468 cell line containing mutant p53. MCF-7 and MDA-MB-468 cells were incubated with different concentrations of ASO or nutlin-3 for various periods of time. VEGF gene and protein expression in tumor cells was measured by qPCR and Western blot. The level of VEGF protein secreted in the culture supernatant of treated cells was quantified by enzyme-linked immunosorbent assay (ELISA). Nude mouse xenograft models were further established to determine their effects on tumor growth and angiogenesis. Serum levels of VEGF were measured by ELISA. VEGF expression and microvessel density in tumor tissues were studied by immunohistochemistry. Both angiogenesis and tumor growth were digitally quantified.

Results

In both MCF-7 and MDA-MB-468 cells, VEGF expression and secretion were reduced, resulting from specific inhibition of MDM2 expression by ASO. In vivo assay, after administration of ASO, VEGF production reduced and anti-angiogenesis activity occurred in nude mice bearing MCF-7 or MDA-MB-468 xenograft. However, in both models treated with nutlin-3, VEGF production was not changed and anti-angiogenesis activity was not observed.

Conclusion

In summary, the ASO construct targeting MDM2 specifically suppresses VEGF expression in vitro and VEGF-mediated tumor angiogenesis in vivo in breast cancer. Furthermore, the suppression of VEGF expression subsequent to inhibition of MDM2 in p53 mutant cells suggests that MDM2 has a regulatory role on VEGF expression through a p53-independent mechanism.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9

References

  1. Schneider BP, Wang M, Radovich M, Sledge GW, Badve S, Thor A, Flockhart DA, Hancock B, Davidson N, Gralow J, Dickler M, Perez EA, Cobleigh M, Shenkier T, Edgerton S, Miller KD, ECOG 2100 (2008) Association of vascular endothelial growth factor and vascular endothelial growth factor receptor-2 genetic polymorphisms with outcome in a trial of paclitaxel compared with paclitaxel plus bevacizumab in advanced breast cancer: ECOG 2100. J Clin Oncol 26(28):4672–4678

    CAS  PubMed  Article  Google Scholar 

  2. Crawford Y, Kasman I, Yu L, Zhong C, Wu X, Modrusan Z, Kaminker J, Ferrara N (2009) PDGF-C mediates the angiogenic and tumorigenic properties of fibroblasts associated with tumors refractory to anti-VEGF treatment. Cancer Cell 15(1):21–34

    CAS  PubMed  Article  Google Scholar 

  3. Maguire M, Nield PC, Devling T, Jenkins RE, Park BK, Polański R, Vlatković N, Boyd MT (2008) MDM2 regulates dihydrofolate reductase activity through monoubiquitination. Cancer Res 68(9):3232–3242

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  4. Turbin DA, Cheang MC, Bajdik CD, Gelmon KA, Yorida E, De Luca A, Nielsen TO, Huntsman DG, Gilks CB (2006) MDM2 protein expression is a negative prognostic marker in breast carcinoma. Mod Pathol 19(1):69–74

    CAS  PubMed  Article  Google Scholar 

  5. Carroll VA, Ashcroft M (2008) Regulation of angiogenic factors by HDM2 in renal cell carcinoma. Cancer Res 68(2):545–552

    CAS  PubMed  Article  Google Scholar 

  6. Zhang Z, Zhang R (2005) p53-independent activities of MDM2 and their relevance to cancer therapy. Curr Cancer Drug Targets 5(1):9–20

    PubMed  Article  Google Scholar 

  7. Mathew R, Arora S, Khanna R, Mathur M, Shukla NK, Ralhan R (2002) Alterations in p53 and pRb pathways and their prognostic significance in oesophageal cancer. Eur J Cancer 38(6):832–841

    CAS  PubMed  Article  Google Scholar 

  8. Momand J, Zambetti GP, Olson DC, George D, Levine AJ (1992) The mdm-2 oncogene product forms a complex with the p53 protein and inhibits p53-mediated transactivation. Cell 69(7):1237–1245

    CAS  PubMed  Article  Google Scholar 

  9. Gu L, Zhu N, Zhang H, Durden DL, Feng Y, Zhou M (2009) Regulation of XIAP translation and induction by MDM2 following irradiation. Cancer Cell 15(5):363–375

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  10. Zhou S, Gu L, He J, Zhang H, Zhou M (2011) MDM2 regulates vascular endothelial growth factor mRNA stabilization in hypoxia. Mol Cell Biol 31(24):4928–4937

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  11. Zhang L, Hill RP (2004) Hypoxia enhances metastatic efficiency by up-regulating Mdm2 in KHT cells and increasing resistance to apoptosis. Cancer Res 64(12):4180–4189

    CAS  PubMed  Article  Google Scholar 

  12. Zietz C, Rössle M, Haas C, Sendelhofert A, Hirschmann A, Stürzl M, Löhrs U (1998) MDM-2 oncoprotein overexpression, p53 gene mutation, and VEGF up-regulation in angiosarcomas. Am J Pathol 153(5):1425–1433

    CAS  PubMed  Article  Google Scholar 

  13. Vassilev LT (2007) MDM2 inhibitors for cancer therapy. Trends Mol Med 13(1):23–31

    CAS  PubMed  Article  Google Scholar 

  14. Tabe Y, Sebasigari D, Jin L, Rudelius M, Davies-Hill T, Miyake K, Miida T, Pittaluga S, Raffeld M (2009) MDM2 antagonist nutlin-3 displays antiproliferative and proapoptotic activity in mantle cell lymphoma. Clin Cancer Res 15(3):933–942

    CAS  PubMed  Article  Google Scholar 

  15. Gu L, Zhu N, Findley HW, Zhou M (2008) MDM2 antagonist nutlin-3 is a potent inducer of apoptosis in pediatric acute lymphoblastic leukemia cells with wild-type p53 and overexpression of MDM2. Leukemia 22(4):730–739

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  16. Zhang R, Wang H, Agrawal S (2005) Novel antisense anti-MDM2 mixed-backbone oligonucleotides: proof of principle, in vitro and in vivo activities, and mechanisms. Curr Cancer Drug Targets 5(1):43–49

    PubMed  Article  Google Scholar 

  17. Zhang Z, Wang H, Prasad G, Li M, Yu D, Bonner JA, Agrawal S, Zhang R (2004) Radiosensitization by antisense anti-MDM2 mixed-backbone oligonucleotide in in vitro and in vivo human cancer models. Clin Cancer Res 10(4):1263–1273

    CAS  PubMed  Article  Google Scholar 

  18. Wang H, Nan L, Yu D, Agrawal S, Zhang R (2001) Antisense anti-MDM2 oligonucleotides as a novel therapeutic approach to human breast cancer: in vitro and in vivo activities and mechanisms. Clin Cancer Res 7(11):3613–3624

    CAS  PubMed  Google Scholar 

  19. Saha MN, Jiang H, Jayakar J, Reece D, Branch DR, Chang H (2010) MDM2 antagonist nutlin plus proteasome inhibitor velcade combination displays a synergistic anti-myeloma activity. Cancer Biol Ther 9(11):936–944

    CAS  PubMed  Article  Google Scholar 

  20. Agrawal S, Jiang Z, Zhao Q, Shaw D, Cai Q, Roskey A, Channavajjala L, Saxinger C, Zhang R (1997) Mixed-backbone oligonucleotides as second generation antisense oligonucleotides: in vitro and in vivo studies. Proc Natl Acad Sci USA 94(6):2620–2625

    CAS  PubMed  Article  Google Scholar 

  21. Vassilev LT, Vu BT, Graves B, Carvajal D, Podlaski F, Filipovic Z, Kong N, Kammlott U, Lukacs C, Klein C, Fotouhi N, Liu EA (2004) In vivo activation of the p53 pathway by small-molecule antagonists of MDM2. Science 303(5659):844–848

    CAS  PubMed  Article  Google Scholar 

  22. Van Maerken T, Ferdinande L, Taildeman J, Lambertz I, Yigit N, Vercruysse L, Rihani A, Michaelis M, Cinatl J Jr, Cuvelier CA, Marine JC, De Paepe A, Bracke M, Speleman F, Vandesompele J (2009) Antitumor activity of the selective MDM2 antagonist nutlin-3 against chemoresistant neuroblastoma with wild-type p53. J Natl Cancer Inst 101(22):1562–1574

    PubMed  Article  Google Scholar 

  23. Bianco R, Caputo R, Caputo R, Damiano V, De Placido S, Ficorella C, Agrawal S, Bianco AR, Ciardiello F, Tortora G (2004) Combined targeting of epidermal growth factor receptor and MDM2 by gefitinib and antisense MDM2 cooperatively inhibit hormone-independent prostate cancer. Clin Cancer Res 10(14):4858–4864

    CAS  PubMed  Article  Google Scholar 

  24. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25(4):402–408

    CAS  PubMed  Article  Google Scholar 

  25. Van Maerken T, Rihani A, Dreidax D, De Clercq S, Yigit N, Marine JC, Westermann F, De Paepe A, Vandesompele J, Speleman F (2011) Functional analysis of the p53 pathway in neuroblastoma cells using the small-molecule MDM2 antagonist nutlin-3. Mol Cancer Ther 10(6):983–993

    PubMed  Article  Google Scholar 

  26. Michael D, Oren M (2002) The p53 and Mdm2 families in cancer. Curr Opin Genet Dev 12(1):53–59

    CAS  PubMed  Article  Google Scholar 

  27. Chène P (2003) Inhibiting the p53-MDM2 interaction: an important target for cancer therapy. Nat Rev Cancer 3(2):102–109

    PubMed  Article  Google Scholar 

  28. Shangary S, Wang S (2008) Targeting the MDM2-p53 interaction for cancer therapy. Clin Cancer Res 14(17):5318–5324

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  29. Mazars R, Spinardi L, BenCheikh M, Simony-Lafontaine J, Jeanteur P, Theillet C (1992) p53 mutations occur in aggressive breast cancer. Cancer Res 52(14):3918–3923

    CAS  PubMed  Google Scholar 

  30. Walerych D, Napoli M, Collavin L, Del Sal G (2012) The rebel angel: mutant p53 as the driving oncogene in breast cancer. Carcinogenesis 33(11):2007–2017

    CAS  PubMed  Article  Google Scholar 

  31. Chen D, Li M, Luo J, Gu W (2003) Direct interactions between HIF-1 alpha and Mdm2 modulate p53 function. J Biol Chem 278(16):13595–13598

    CAS  PubMed  Article  Google Scholar 

  32. Nieminen AL, Qanungo S, Schneider EA, Jiang BH, Agani FH (2005) Mdm2 and HIF-1alpha interaction in tumor cells during hypoxia. J Cell Physiol 204(2):364–369

    CAS  PubMed  Article  Google Scholar 

  33. LaRusch GA, Jackson MW, Dunbar JD, Warren RS, Donner DB, Mayo LD (2007) Nutlin3 blocks vascular endothelial growth factor induction by preventing the interaction between hypoxia inducible factor 1alpha and Hdm2. Cancer Res 67(2):450–454

    CAS  PubMed  Article  Google Scholar 

  34. Lee YM, Lim JH, Chun YS, Moon HE, Lee MK, Huang LE, Park JW (2009) Nutlin-3, an Hdm2 antagonist, inhibits tumor adaptation to hypoxia by stimulating the FIH-mediated inactivation of HIF-1alpha. Carcinogenesis 30(10):1768–1775

    CAS  PubMed  Article  Google Scholar 

  35. Patterson DM, Gao D, Trahan DN, Johnson BA, Ludwig A, Barbieri E, Chen Z, Diaz-Miron J, Vassilev L, Shohet JM, Kim ES (2011) Effect of MDM2 and vascular endothelial growth factor inhibition on tumor angiogenesis and metastasis in neuroblastoma. Angiogenesis 14(3):255–266

    CAS  PubMed  Article  Google Scholar 

  36. Levy AP, Levy NS, Goldberg MA (1996) Post-transcriptional regulation of vascular endothelial growth factor by hypoxia. J Biol Chem 271(5):2746–2753

    CAS  PubMed  Article  Google Scholar 

  37. Shih SC, Claffey KP (1999) Regulation of human vascular endothelial growth factor mRNA stability in hypoxia by heterogeneous nuclear ribonucleoprotein L. J Biol Chem 274(3):1359–1365

    CAS  PubMed  Article  Google Scholar 

  38. Goldberg-Cohen I, Furneauxb H, Levy AP (2002) A 40-bp RNA element that mediates stabilization of vascular endothelial growth factor mRNA by HuR. J Biol Chem 277(16):13635–13640

    CAS  PubMed  Article  Google Scholar 

  39. Vumbaca F, Phoenix KN, Rodriguez-Pinto D, Han DK, Claffey KP (2008) Double-stranded RNA-binding protein regulates vascular endothelial growth factor mRNA stability, translation, and breast cancer angiogenesis. Mol Cell Biol 28(2):772–783

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  40. Tordai A, Wang J, Andre F, Liedtke C, Yan K, Sotiriou C, Hortobagyi GN, Symmans WF, Pusztai L (2008) Evaluation of biological pathways involved in chemotherapy response in breast cancer. Breast Cancer Res 10(2):R37

    PubMed Central  PubMed  Article  Google Scholar 

  41. Lu X, Liu DP, Xu Y (2013) The gain of function of p53 cancer mutant in promoting mammary tumorigenesis. Oncogene 32(23):2900–2906

    CAS  PubMed Central  PubMed  Article  Google Scholar 

Download references

Acknowledgments

This work was supported by National Natural Science Foundation of China Grant 81072168.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Sheng Zhou.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Xiong, J., Yang, Q., Li, J. et al. Effects of MDM2 inhibitors on vascular endothelial growth factor-mediated tumor angiogenesis in human breast cancer. Angiogenesis 17, 37–50 (2014). https://doi.org/10.1007/s10456-013-9376-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10456-013-9376-3

Keywords

  • Breast neoplasms
  • Angiogenesis
  • VEGF
  • MDM2
  • Antisense oligonucleotide