The oncoprotein HBXIP promotes human breast cancer growth through down-regulating p53 via miR-18b/MDM2 and pAKT/MDM2 pathways

Abstract

Mammalian hepatitis B X-interacting protein (HBXIP) is an 18-kDa protein that regulates a large number of transcription factors such as TF-IID, E2F1, SP1, STAT3, c-Myc, and LXR by serving as an oncogenic transcription coactivator and plays an important role in the development of breast cancer. We previously showed that HBXIP as an oncoprotein could enhance the promoter activity of MDM2 through coactivating p53, promoting the MDM2 transcription in breast cancer. In this study we investigated the molecular mechanisms underlying the modulation of MDM2/p53 interaction by HBXIP in human breast cancer MCF-7 cells in vitro and in vivo. We showed that HBXIP could up-regulate MDM2 through inducing DNA methylation of miR-18b, thus suppressing the miR-18b expression, leading to the attenuation of p53 in breast cancer cells. In addition, HBXIP could promote the phosphorylation of MDM2 by increasing the level of pAKT and bind to pMDM2, subsequently enhancing the interaction between MDM2 and p53 for the down-regulation of p53 in breast cancer cells. In MCF-7 breast cancer xenograft nude mice, we also observed that overexpression of HBXIP promoted breast cancer growth through the miR-18b/MDM2 and pAKT/MDM2 pathways. In conclusion, oncoprotein HBXIP suppresses miR-18b to elevate MDM2 and activates pAKT to phosphorylate MDM2 for enhancing the interaction between MDM2 and p53, leading to p53 degradation in promotion of breast cancer growth. Our findings shed light on a novel mechanism of p53 down-regulation during the development of breast cancer.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

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

References

  1. 1.

    Wang L, Wu Q, Qiu P, Mirza A, McGuirk M, Kirschmeier P, et al. Analyses of p53 target genes in the human genome by bioinformatic and microarray approaches. J Biol Chem. 2001;276:43604–10.

  2. 2.

    Mandriani B, Castellana S, Rinaldi C, Manzoni M, Venuto S, Rodriguez-Aznar E, et al. Identification of p53-target genes in Danio rerio. Sci Rep. 2016;6:32474.

  3. 3.

    Yao GD, Yang J, Li Q, Zhang Y, Qi M, Fan SM, et al. Activation of p53 contributes to pseudolaric acid B-induced senescence in human lung cancer cells in vitro. Acta Pharmacol Sin. 2016;37:919–29.

  4. 4.

    Amaral JD, Castro RE, Sola S, Steer CJ, Rodrigues CM. p53 is a key molecular target of ursodeoxycholic acid in regulating apoptosis. J Biol Chem. 2007;282:34250–9.

  5. 5.

    Wiegering A, Matthes N, Muhling B, Koospal M, Quenzer A, Peter S, et al. Reactivating p53 and inducing tumor apoptosis (RITA) enhances the response of RITA-sensitive colorectal cancer cells to chemotherapeutic agents 5-fluorouracil and oxaliplatin. Neoplasia. 2017;19:301–9.

  6. 6.

    Su LY, Shi YX, Yan MR, Xi Y, Su XL. Anticancer bioactive peptides suppress human colorectal tumor cell growth and induce apoptosis via modulating the PARP-p53-Mcl-1 signaling pathway. Acta Pharmacol Sin. 2015;36:1514–9.

  7. 7.

    Hollstein M, Hainaut P. Massively regulated genes: the example of TP53. J Pathol. 2010;220:164–73.

  8. 8.

    Feng FY, Zhang Y, Kothari V, Evans JR, Jackson WC, Chen W, et al. MDM2 inhibition sensitizes prostate cancer cells to androgen ablation and radiotherapy in a p53-dependent manner. Neoplasia. 2016;18:213–22.

  9. 9.

    Momand J, Jung D, Wilczynski S, Niland J. The MDM2 gene amplification database. Nucl Acids Res. 1998;26:3453–9.

  10. 10.

    Thomasova D, Mulay SR, Bruns H, Anders HJ. p53-independent roles of MDM2 in NF-kappaB signaling: implications for cancer therapy, wound healing, and autoimmune diseases. Neoplasia. 2012;14:1097–101.

  11. 11.

    Kubbutat MH, Jones SN, Vousden KH. Regulation of p53 stability by Mdm2. Nature. 1997;387:299–303.

  12. 12.

    Sczaniecka M, Gladstone K, Pettersson S, McLaren L, Huart AS, Wallace M. MDM2 protein-mediated ubiquitination of numb protein: identification of a second physiological substrate of MDM2 that employs a dual-site docking mechanism. J Biol Chem. 2012;287:14052–68.

  13. 13.

    Honda R, Tanaka H, Yasuda H. Oncoprotein MDM2 is a ubiquitin ligase E3 for tumor suppressor p53. FEBS Lett. 1997;420:25–7.

  14. 14.

    Ciemny MP, Debinski A, Paczkowska M, Kolinski A, Kurcinski M, Kmiecik S. Protein-peptide molecular docking with large-scale conformational changes: the p53–MDM2 interaction. Sci Rep. 2016;6:37532.

  15. 15.

    Phelps M, Darley M, Primrose JN, Blaydes JP. p53-independent activation of the hdm2–P2 promoter through multiple transcription factor response elements results in elevated hdm2 expression in estrogen receptor alpha-positive breast cancer cells. Cancer Res. 2003;63:2616–23.

  16. 16.

    Dai MS, Lu H. Inhibition of MDM2-mediated p53 ubiquitination and degradation by ribosomal protein L5. J Biol Chem. 2004;279:44475–82.

  17. 17.

    Dar AA, Majid S, Rittsteuer C, de Semir D, Bezrookove V, Tong S, et al. The role of miR-18b in MDM2-p53 pathway signaling and melanoma progression. J Natl Cancer Inst. 2013;105:433–42.

  18. 18.

    Mendell JT. miRiad roles for the miR-17-92 cluster in development and disease. Cell. 2008;133:217–22.

  19. 19.

    Tatsuguchi M, Seok HY, Callis TE, Thomson JM, Chen JF, Newman M, et al. Expression of microRNAs is dynamically regulated during cardiomyocyte hypertrophy. J Mol Cell Cardiol. 2007;42:1137–41.

  20. 20.

    Zhang ZZ, Liu X, Wang DQ, Teng MK, Niu LW, Huang AL, et al. Hepatitis B virus and hepatocellular carcinoma at the miRNA level. World J Gastroenterol. 2011;17:3353–8.

  21. 21.

    Leivonen SK, Makela R, Ostling P, Kohonen P, Haapa-Paananen S, Kleivi K, et al. Protein lysate microarray analysis to identify microRNAs regulating estrogen receptor signaling in breast cancer cell lines. Oncogene. 2009;28:3926–36.

  22. 22.

    Maya R, Balass M, Kim ST, Shkedy D, Leal JF, Shifman O, et al. ATM-dependent phosphorylation of Mdm2 on serine 395: role in p53 activation by DNA damage. Genes Dev. 2001;15:1067–77.

  23. 23.

    Carr MI, Roderick JE, Zhang H, Woda BA, Kelliher MA, Jones SN. Phosphorylation of the Mdm2 oncoprotein by the c-Abl tyrosine kinase regulates p53 tumor suppression and the radiosensitivity of mice. Proc Natl Acad Sci USA. 2016;113:15024–9.

  24. 24.

    Ogawara Y, Kishishita S, Obata T, Isazawa Y, Suzuki T, Tanaka K, et al. Akt enhances Mdm2-mediated ubiquitination and degradation of p53. J Biol Chem. 2002;277:21843–50.

  25. 25.

    Bar-Peled L, Schweitzer LD, Zoncu R, Sabatini DM. Ragulator is a GEF for the rag GTPases that signal amino acid levels to mTORC1. Cell. 2012;150:1196–208.

  26. 26.

    Melegari M, Scaglioni PP, Wands JR. Cloning and characterization of a novel hepatitis B virus x binding protein that inhibits viral replication. J Virol. 1998;72:1737–43.

  27. 27.

    Fujii R, Zhu C, Wen Y, Marusawa H, Bailly-Maitre B, Matsuzawa S, et al. HBXIP, cellular target of hepatitis B virus oncoprotein, is a regulator of centrosome dynamics and cytokinesis. Cancer Res. 2006;66:9099–107.

  28. 28.

    Li H, Liu Q, Wang Z, Fang R, Shen Y, Cai X, et al. The oncoprotein HBXIP modulates the feedback loop of MDM2/p53 to enhance the growth of breast cancer. J Biol Chem. 2015;290:22649–61.

  29. 29.

    Liu Q, Bai X, Li H, Zhang Y, Zhao Y, Zhang X, et al. The oncoprotein HBXIP upregulates Lin28B via activating TF II D to promote proliferation of breast cancer cells. Int J Cancer. 2013;133:1310–22.

  30. 30.

    Liu F, You X, Wang Y, Liu Q, Liu Y, Zhang S, et al. The oncoprotein HBXIP enhances angiogenesis and growth of breast cancer through modulating FGF8 and VEGF. Carcinogenesis. 2014;35:1144–53.

  31. 31.

    Li Y, Wang Z, Shi H, Li H, Li L, Fang R, et al. HBXIP and LSD1 scaffolded by lncRNA hotair mediate transcriptional activation by c-Myc. Cancer Res. 2016;76:293–304.

  32. 32.

    Zhao Y, Li H, Zhang Y, Li L, Fang R, Li Y, et al. Oncoprotein HBXIP modulates abnormal lipid metabolism and growth of breast cancer cells by activating the LXRs/SREBP-1c/FAS signaling cascade. Cancer Res. 2016;76:4696–707.

  33. 33.

    Zhou XL, Guo X, Song YP, Zhu CY, Zou W. The LPI/GPR55 axis enhances human breast cancer cell migration via HBXIP and p-MLC signaling. Acta Pharmacol Sin. 2018;39:459–71.

  34. 34.

    Hu N, Zhang J, Cui W, Kong G, Zhang S, Yue L, et al. miR-520b regulates migration of breast cancer cells by targeting hepatitis B X-interacting protein and interleukin-8. J Biol Chem. 2011;286:13714–22.

  35. 35.

    Shi X, Kachirskaia I, Yamaguchi H, West LE, Wen H, Wang EW, et al. Modulation of p53 function by SET8-mediated methylation at lysine 382. Mol Cell. 2007;27:636–46.

  36. 36.

    Han S, Khuri FR, Roman J. Fibronectin stimulates non-small cell lung carcinoma cell growth through activation of Akt/mammalian target of rapamycin/S6 kinase and inactivation of LKB1/AMP-activated protein kinase signal pathways. Cancer Res. 2006;66:315–23.

  37. 37.

    Marusawa H, Matsuzawa S, Welsh K, Zou H, Armstrong R, Tamm I, et al. HBXIP functions as a cofactor of survivin in apoptosis suppression. EMBO J. 2003;22:2729–40.

  38. 38.

    Zhang Y, Zhao Y, Li H, Li Y, Cai X, Shen Y, et al. The nuclear import of oncoprotein hepatitis B X-interacting protein depends on interacting with c-Fos and phosphorylation of both proteins in breast cancer cells. J Biol Chem. 2013;288:18961–74.

  39. 39.

    Shan C, Xu F, Zhang S, You J, You X, Qiu L, et al. Hepatitis B virus X protein promotes liver cell proliferation via a positive cascade loop involving arachidonic acid metabolism and p-ERK1/2. Cell Res. 2010;20:563–75.

  40. 40.

    Gonzalez L, Agullo-Ortuno MT, Garcia-Martinez JM, Calcabrini A, Gamallo C, Palacios J, et al. Role of c-Src in human MCF7 breast cancer cell tumorigenesis. J Biol Chem. 2006;281:20851–64.

  41. 41.

    Yu X, Zhen Y, Yang H, Wang H, Zhou Y, Wang E, et al. Loss of connective tissue growth factor as an unfavorable prognosis factor activates miR-18b by PI3K/AKT/C-Jun and C-Myc and promotes cell growth in nasopharyngeal carcinoma. Cell Death Dis. 2013;4:e634.

  42. 42.

    Li LC, Dahiya R. MethPrimer: designing primers for methylation PCRs. Bioinformatics. 2002;18:1427–31.

  43. 43.

    von der Chevallerie K, Rolfes S, Schierwater B. Inhibitors of the p53-Mdm2 interaction increase programmed cell death and produce abnormal phenotypes in the placozoon Trichoplax adhaerens (F.E. Schulze). Dev Genes Evol. 2014;224:79–85.

  44. 44.

    Touqan N, Diggle CP, Verghese ET, Perry S, Horgan K, Merchant W, et al. An observational study on the expression levels of MDM2 and MDMX proteins, and associated effects on P53 in a series of human liposarcomas. BMC Clin Pathol. 2013;13:32.

  45. 45.

    Wang X, Wang J, Jiang X. MdmX protein is essential for Mdm2 protein-mediated p53 polyubiquitination. J Biol Chem. 2011;286:23725–34.

  46. 46.

    Liu S, Li L, Zhang Y, Zhang Y, Zhao Y, You X, et al. The oncoprotein HBXIP uses two pathways to up-regulate S100A4 in promotion of growth and migration of breast cancer cells. J Biol Chem. 2012;287:30228–39.

  47. 47.

    Fischer M, Steiner L, Engeland K. The transcription factorp53: not a repressor, solely an activator. Cell Cycle. 2014;13:3037–58.

  48. 48.

    Hayashi Y, Tsujii M, Kodama T, Akasaka T, Kondo J, Hikita H, et al. p53 functional deficiency in human colon cancer cells promotes fibroblast-mediated angiogenesis and tumor growth. Carcinogenesis. 2016;37:972–84.

  49. 49.

    He Y, Lian G, Lin S, Ye Z, Li Q. MDM2 inhibits axin-induced p53 activation independently of its E3 ligase activity. PLoS ONE. 2013;8:e67529.

  50. 50.

    Jazirehi AR, Torres-Collado AX, Nazarian R. Role of miR-18b/MDM2/p53 circuitry in melanoma progression. Epigenomics. 2013;5:254.

  51. 51.

    Yoshimoto N, Toyama T, Takahashi S, Sugiura H, Endo Y, Iwasa M, et al. Distinct expressions of microRNAs that directly target estrogen receptor alpha in human breast cancer. Breast Cancer Res Treat. 2011;130:331–9.

  52. 52.

    Fonseca-Sanchez MA, Perez-Plasencia C, Fernandez-Retana J, Arechaga-Ocampo E, Marchat LA, Rodriguez-Cuevas S, et al. MicroRNA-18b is upregulated in breast cancer and modulates genes involved in cell migration. Oncol Rep. 2013;30:2399–410.

  53. 53.

    Stork PJ, Schmitt JM. Crosstalk between cAMP and MAP kinase signaling in the regulation of cell proliferation. Trends Cell Biol. 2002;12:258–66.

  54. 54.

    Macneil LT, Walhout AJ. Gene regulatory networks and the role of robustness and stochasticity in the control of gene expression. Genome Res. 2011;21:645–57.

  55. 55.

    Xia L, Paik A, Li JJ. p53 activation in chronic radiation-treated breast cancer cells: regulation of MDM2/p14ARF. Cancer Res. 2004;64:221–8.

  56. 56.

    Wu CT, Lin TY, Hsu HY, Sheu F, Ho CM, Chen EI. Ling Zhi-8 mediates p53-dependent growth arrest of lung cancer cells proliferation via the ribosomal protein S7-MDM2-p53 pathway. Carcinogenesis. 2011;32:1890–6.

  57. 57.

    Michael D, Oren M. The p53-Mdm2 module and the ubiquitin system. Semin Cancer Biol. 2003;13:49–58.

Download references

Acknowledgements

This work was supported by grants from the National Basic Research Program of China (973 Program No. 2015CB553905), the National Natural Scientific Foundation of China (Nos. 81372186, 31670771), the Fundamental Research Funds for the Central Universities, Project of Prevention and Control of Key Chronic Non Infectious Diseases (No. 2016YFC1303401), CAMS Innovation Fund for Medical Sciences (CIFMS, 2017-I2M-3-019 and 2016-I2M-1-017), the PUMC Youth Fund and the Fundamental Research Funds for the Central Universities (No. 2017310027), the Tianjin Science and Technology Support Plan Project (TJKJZC, 14ZCZDSY00001).

Author contributions

HL, ZW, MJ, YS, X-lC, and QL performed the experiments. HL, R-pF, HS, and KY carried out the data analysis. HL, S-jF, W-yZ, L-hY designed the study. S-jF, W-yZ, and L-hY supervised the study. HL, S-jF, W-yZ, and L-hY wrote the manuscript with input from all authors.

Author information

Correspondence to Sai-jun Fan or Wei-ying Zhang or Li-hong Ye.

Ethics declarations

Competing interests

The authors declare no competing interests.

Electronic supplementary material

Supplementary Information

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Keywords

  • HBXIP
  • p53
  • MDM2
  • miR-18b
  • pAKT
  • human breast cancer
  • cell proliferation
  • MCF-7 cells
  • cancer xenograft nude mice.

Further reading