Advertisement

Journal of Cancer Research and Clinical Oncology

, Volume 144, Issue 7, pp 1289–1300 | Cite as

Mycoplasma hyorhinis reduces sensitivity of human lung carcinoma cells to Nutlin-3 and promotes their malignant phenotype

  • Uljana A. Boyarskikh
  • Alexandra S. Shadrina
  • Mariya A. Smetanina
  • Yakov A. Tsepilov
  • Igor P. Oscorbin
  • Vadim V. Kozlov
  • Alexander E. Kel
  • Maxim L. Filipenko
Original Article – Cancer Research

Abstract

Purpose

MDM2 inhibitors are promising anticancer agents that induce cell cycle arrest and tumor cells death via p53 reactivation. We examined the influence of Mycoplasma hyorhinis infection on sensitivity of human lung carcinoma cells NCI-H292 to MDM2 inhibitor Nutlin-3. In order to unveil possible mechanisms underlying the revealed effect, we investigated gene expression changes and signal transduction networks activated in NCI-H292 cells in response to mycoplasma infection.

Methods

Sensitivity of NCI-Н292 cells to Nutlin-3 was estimated by resazurin-based cell viability assay. Genome-wide transcriptional profiles of NCI-H292 and NCI-Н292Myc.h cell lines were determined using Illumina Human HT-12 v3 Expression BeadChip. Search for key transcription factors and key node molecules was performed using the geneXplain platform. Ability for anchorage-independent growth was tested by soft agar colony formation assay.

Results

NCI-Н292Myc.h cells were shown to be 1.5- and 5.2-fold more resistant to killing by Nutlin-3 at concentrations of 15 and 30 µM than uninfected NCI-Н292 cells (P < 0.05 and P < 0.001, respectively). Transcriptome analysis revealed differential expression of multiple genes involved in cancer progression and metastasis as well as epithelial–mesenchymal transition (EMT). Moreover, we have shown experimentally that NCI-Н292Myc.h cells were more capable of growing and dividing without binding to a substrate. The most likely mechanism explaining the observed changes was found to be TLR4- and IL-1b-mediated activation of NF-κB pathway.

Conclusions

Our results provide evidence that mycoplasma infection is an important factor modulating the effect of MDM2 inhibitors on cancer cells and is able to induce EMT-related changes.

Keywords

Mycoplasma Lung cancer MDM2 inhibitor Nutlin-3 NF-κB Epithelial–mesenchymal transition 

Notes

Acknowledgements

We thank Dr. Olga Timofeeva (Georgetown University Medical Center, USA) who kindly provided NCI-H292 cell line infected with M. hyorhinis (Н292Myc.h).

Funding

This work was supported by the Federal Targeted Program “Research and development on priority directions of science and technology in Russia, 2014–2020”, contract № 14.604.21.0101, unique identifier of the applied scientific project: RFMEFI60414X0101.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Informed consent

Informed consent was obtained from all individual participants included in the study.

Supplementary material

432_2018_2658_MOESM1_ESM.xlsx (35 kb)
Supplementary material 1 (XLSX 35 KB)
432_2018_2658_MOESM2_ESM.docx (56 kb)
Supplementary material 2 (DOCX 55 KB)
432_2018_2658_MOESM3_ESM.xlsx (22 kb)
Supplementary material 3 (XLSX 22 KB)
432_2018_2658_MOESM4_ESM.xlsx (11 kb)
Supplementary material 4 (XLSX 10 KB)
432_2018_2658_MOESM5_ESM.xlsx (11 kb)
Supplementary material 5 (XLSX 10 KB)
432_2018_2658_MOESM6_ESM.xlsx (12 kb)
Supplementary material 6 (XLSX 11 KB)
432_2018_2658_MOESM7_ESM.jpg (1.6 mb)
Online fig. 1 Visualization of gene expression data by means of heatmap. Up-regulated genes are shown in red. Down-regulated genes are shown in blue. Н292_M1 and Н292_M2 – Н292 cells infected with M. hyorhinis; Н292_C1 and Н292_C2 – mycoplasma-free Н292 cells (JPG 1608 KB)
432_2018_2658_MOESM8_ESM.jpg (2.3 mb)
Online fig. 2 Results of a key node analysis. Summary network of signaling cascades activated in NCI-H292 cells infected with M. hyorhinis. Key node molecules and their complexes are shown in pink. Up-regulated transcription factors are shown in blue. Yellow frame indicates an increased level of expression of one or more molecules in a complex (JPG 2387 KB)
432_2018_2658_MOESM9_ESM.jpg (464 kb)
Online fig. 3 IL-1b-mediated positive feedback loop on a scheme generated by a key node analysis. Key node molecules and their complexes are shown in pink. Up-regulated transcription factors are shown in blue. Yellow frame indicates an increased level of expression of one or more molecules in a complex (JPG 463 KB)

References

  1. Apostolou P, Tsantsaridou A, Papasotiriou I et al (2011) Bacterial and fungal microflora in surgically removed lung cancer samples. J Cardiothorac Surg 6:137.  https://doi.org/10.1186/1749-8090-6-137 CrossRefPubMedPubMedCentralGoogle Scholar
  2. Bian B, Mongrain S, Cagnol S et al (2016) Cathepsin B promotes colorectal tumorigenesis, cell invasion, and metastasis. Mol Carcinog 55:671–687.  https://doi.org/10.1002/mc.22312 CrossRefPubMedGoogle Scholar
  3. Bugide S, Gonugunta VK, Penugurti V et al (2017) HPIP promotes epithelial–mesenchymal transition and cisplatin resistance in ovarian cancer cells through PI3K/AKT pathway activation. Cell Oncol 40:133–144.  https://doi.org/10.1007/s13402-016-0308-2 CrossRefGoogle Scholar
  4. Burgess A, Chia KM, Haupt S et al (2016) Clinical overview of MDM2/X-targeted therapies. Front Oncol 6:7.  https://doi.org/10.3389/fonc.2016.00007 CrossRefPubMedPubMedCentralGoogle Scholar
  5. Cao C, Shinohara ET, Subhawong TK et al (2006) Radiosensitization of lung cancer by nutlin, an inhibitor of murine double minute 2. Mol Cancer Ther 5:411–417.  https://doi.org/10.1158/1535-7163.MCT-05-0356 CrossRefPubMedGoogle Scholar
  6. Cogswell JP, Godlevski MM, Wisely GB et al (1994) NF-kappa B regulates IL-1 beta transcription through a consensus NF-kappa B binding site and a nonconsensus CRE-like site. J Immunol 153:712–723PubMedGoogle Scholar
  7. Deben C, Wouters A, Beeck K, Op de et al (2015) The MDM2-inhibitor Nutlin-3 synergizes with cisplatin to induce p53 dependent tumor cell apoptosis in non-small cell lung cancer. Oncotarget 6:22666–22679.  https://doi.org/10.18632/oncotarget.4433 CrossRefPubMedPubMedCentralGoogle Scholar
  8. Deben C, Deschoolmeester V, Lardon F et al (2016) TP53 and MDM2 genetic alterations in non-small cell lung cancer: evaluating their prognostic and predictive value. Crit Rev Oncol Hematol 99:63–73.  https://doi.org/10.1016/j.critrevonc.2015.11.019 CrossRefPubMedGoogle Scholar
  9. Dey A, Wong ET, Bist P et al (2007) Nutlin-3 inhibits the NFκB pathway in a p53 dependent manner: implications in lung cancer therapy. Cell Cycle 6:2178–2185.  https://doi.org/10.4161/cc.6.17.4643 CrossRefPubMedGoogle Scholar
  10. Donovan ML, Schultz TE, Duke TJ, Blumenthal A (2017) Type I interferons in the pathogenesis of tuberculosis: molecular drivers and immunological consequences. Front Immunol 8:1633.  https://doi.org/10.3389/fimmu.2017.01633 CrossRefPubMedPubMedCentralGoogle Scholar
  11. Duan H, Chen L, Qu L et al (2014) Mycoplasma hyorhinis infection promotes NF-kB-dependent migration of gastric cancer cells. Cancer Res 74:5782–5794.  https://doi.org/10.1158/0008-5472.CAN-14-0650 CrossRefPubMedGoogle Scholar
  12. Dziarski R, Gupta D (2000) Role of MD-2 in TLR2- and TLR4-mediated recognition of Gram-negative and Gram-positive bacteria and activation of chemokine genes. J Endotoxin Res 6:401–405CrossRefPubMedGoogle Scholar
  13. Edge SB, Compton CC (2010) The American Joint Committee on Cancer: the 7th Edition of the AJCC cancer staging manual and the future of TNM. Ann Surg Oncol 17:1471–1474.  https://doi.org/10.1245/s10434-010-0985-4 CrossRefPubMedGoogle Scholar
  14. Feng SH, Tsai S, Rodriguez J, Lo SC (1999) Mycoplasmal infections prevent apoptosis and induce malignant transformation of interleukin-3-dependent 32D hematopoietic cells. Mol Cell Biol 19:7995–8002CrossRefPubMedPubMedCentralGoogle Scholar
  15. Geller LT, Barzily-Rokni M, Danino T et al (2017) Potential role of intratumor bacteria in mediating tumor resistance to the chemotherapeutic drug gemcitabine. Science 357:1156–1160.  https://doi.org/10.1126/science.aah5043 CrossRefPubMedPubMedCentralGoogle Scholar
  16. Gomersall AC, Phan HA, Iacuone S et al (2015) The Mycoplasma hyorhinis p37 protein rapidly induces genes in fibroblasts associated with inflammation and cancer. PLoS One 10:e0140753.  https://doi.org/10.1371/journal.pone.0140753 CrossRefPubMedPubMedCentralGoogle Scholar
  17. Gong M, Meng L, Jiang B et al (2008) p37 from Mycoplasma hyorhinis promotes cancer cell invasiveness and metastasis through activation of MMP-2 and followed by phosphorylation of EGFR. Mol Cancer Ther 7:530–537.  https://doi.org/10.1158/1535-7163.MCT-07-2191 CrossRefPubMedGoogle Scholar
  18. Goodison S, Nakamura K, Iczkowski KA et al (2007) Exogenous mycoplasmal p37 protein alters gene expression, growth and morphology of prostate cancer cells. Cytogenet Genome Res 118:204–213.  https://doi.org/10.1159/000108302 CrossRefPubMedGoogle Scholar
  19. Griner SE, Joshi JP, Nahta R (2013) Growth differentiation factor 15 stimulates rapamycin-sensitive ovarian cancer cell growth and invasion. Biochem Pharmacol 85:46–58.  https://doi.org/10.1016/j.bcp.2012.10.007 CrossRefPubMedGoogle Scholar
  20. Hai J, Sakashita S, Allo G et al (2015) Inhibiting MDM2-p53 interaction suppresses tumor growth in patient-derived non-small cell lung cancer xenograft models. J Thorac Oncol 10:1172–1180.  https://doi.org/10.1097/JTO.0000000000000584 CrossRefPubMedGoogle Scholar
  21. Huang S, Li JY, Wu J et al (2001) Mycoplasma infections and different human carcinomas. World J Gastroenterol 7:266–269.  https://doi.org/10.3748/wjg.v7.i2.266 CrossRefPubMedPubMedCentralGoogle Scholar
  22. Iida N, Dzutsev A, Stewart CA et al (2013) Commensal bacteria control cancer response to therapy by modulating the tumor microenvironment. Science 342:967–970.  https://doi.org/10.1126/science.1240527 CrossRefPubMedGoogle Scholar
  23. Jetté L, Bissoon-Haqqani S, Le François B et al (2008) Resistance of colorectal cancer cells to 5-FUdR and 5-FU caused by Mycoplasma infection. Anticancer Res 28:2175–2180PubMedGoogle Scholar
  24. Jiang S, Zhang S, Langenfeld J et al (2008) Mycoplasma infection transforms normal lung cells and induces bone morphogenetic protein 2 expression by post-transcriptional mechanisms. J Cell Biochem 104:580–594.  https://doi.org/10.1002/jcb.21647 CrossRefPubMedGoogle Scholar
  25. Karin M, Lin A (2002) NF-κB at the crossroads of life and death. Nat Immunol 3:221–227.  https://doi.org/10.1038/ni0302-221 CrossRefPubMedGoogle Scholar
  26. Kawasaki T, Kawai T (2014) Toll-like receptor signaling pathways. Front Immunol 5:461.  https://doi.org/10.3389/fimmu.2014.00461 CrossRefPubMedPubMedCentralGoogle Scholar
  27. Kegelman TP, Das SK, Emdad L et al (2015) Targeting tumor invasion: the roles of MDA-9/Syntenin. Expert Opin Ther Targets 19:97–112.  https://doi.org/10.1517/14728222.2014.959495 CrossRefPubMedGoogle Scholar
  28. Kel A, Voss N, Jauregui R et al (2006) Beyond microarrays: find key transcription factors controlling signal transduction pathways. BMC Bioinform 7(Suppl 2):S13.  https://doi.org/10.1186/1471-2105-7-S2-S13 CrossRefGoogle Scholar
  29. Ketcham CM, Anai S, Reutzel R et al (2005) p37 Induces tumor invasiveness. Mol Cancer Ther 4:1031–1038.  https://doi.org/10.1158/1535-7163.MCT-05-0040 CrossRefPubMedGoogle Scholar
  30. Killip MJ, Fodor E, Randall RE (2015) Influenza virus activation of the interferon system. Virus Res 209:11–22.  https://doi.org/10.1016/j.virusres.2015.02.003 CrossRefPubMedPubMedCentralGoogle Scholar
  31. Krull M, Pistor S, Voss N et al (2006) TRANSPATH: an information resource for storing and visualizing signaling pathways and their pathological aberrations. Nucleic Acids Res 34:D546–D551.  https://doi.org/10.1093/nar/gkj107 CrossRefPubMedGoogle Scholar
  32. Lai J-F, Zindl CL, Duffy LB et al (2010) Critical role of macrophages and their activation via MyD88-NFκB signaling in lung innate immunity to Mycoplasma pneumoniae. PLoS One 5:e14417.  https://doi.org/10.1371/journal.pone.0014417 CrossRefPubMedPubMedCentralGoogle Scholar
  33. Lamouille S, Xu J, Derynck R (2014) Molecular mechanisms of epithelial–mesenchymal transition. Nat Rev Mol Cell Biol 15:178–196.  https://doi.org/10.1038/nrm3758 CrossRefPubMedPubMedCentralGoogle Scholar
  34. Lawrence T (2009) The nuclear factor NF-kappaB pathway in inflammation. Cold Spring Harb Perspect Biol 1:a001651.  https://doi.org/10.1101/cshperspect.a001651 CrossRefPubMedPubMedCentralGoogle Scholar
  35. Li C, Wang J, Kong J et al (2016) GDF15 promotes EMT and metastasis in colorectal cancer. Oncotarget 7:860–872.  https://doi.org/10.18632/oncotarget.6205 PubMedGoogle Scholar
  36. Li C, Zhang J, Wu H et al (2017) Lectin-like oxidized low-density lipoprotein receptor-1 facilitates metastasis of gastric cancer through driving epithelial–mesenchymal transition and PI3K/Akt/GSK3β activation. Sci Rep 7:45275.  https://doi.org/10.1038/srep45275 CrossRefPubMedPubMedCentralGoogle Scholar
  37. Liekens S, Bronckaers A, Balzarini J (2009) Improvement of purine and pyrimidine antimetabolite-based anticancer treatment by selective suppression of mycoplasma-encoded catabolic enzymes. Lancet Oncol 10:628–635.  https://doi.org/10.1016/S1470-2045(09)70037-3 CrossRefPubMedGoogle Scholar
  38. Liu L, Zhou X-M, Yang F-F et al (2017) TRIM22 confers poor prognosis and promotes epithelial–mesenchymal transition through regulation of AKT/GSK3β/β-catenin signaling in non-small cell lung cancer. Oncotarget 8:62069–62080.  https://doi.org/10.18632/oncotarget.18911 PubMedPubMedCentralGoogle Scholar
  39. Logunov DY, Scheblyakov DV, Zubkova OV et al (2008) Mycoplasma infection suppresses p53, activates NF-kappaB and cooperates with oncogenic Ras in rodent fibroblast transformation. Oncogene 27:4521–4531.  https://doi.org/10.1038/onc.2008.103 CrossRefPubMedPubMedCentralGoogle Scholar
  40. Matys V, Kel-Margoulis OV, Fricke E et al (2006) TRANSFAC and its module TRANSCompel: transcriptional gene regulation in eukaryotes. Nucleic Acids Res 34:D108–D110.  https://doi.org/10.1093/nar/gkj143 CrossRefPubMedGoogle Scholar
  41. Mi H, Muruganujan A, Casagrande JT, Thomas PD (2013) Large-scale gene function analysis with the PANTHER classification system. Nat Protoc 8:1551–1566.  https://doi.org/10.1038/nprot.2013.092 CrossRefPubMedGoogle Scholar
  42. Mitrović A, Pečar Fonović U, Kos J (2017) Cysteine cathepsins B and X promote epithelial–mesenchymal transition of tumor cells. Eur J Cell Biol 96:622–631.  https://doi.org/10.1016/j.ejcb.2017.04.003 CrossRefPubMedGoogle Scholar
  43. Mori S, Chang JT, Andrechek ER et al (2009) Anchorage-independent cell growth signature identifies tumors with metastatic potential. Oncogene 28:2796–2805.  https://doi.org/10.1038/onc.2009.139 CrossRefPubMedPubMedCentralGoogle Scholar
  44. Namiki K, Goodison S, Porvasnik S et al (2009) Persistent exposure to Mycoplasma induces malignant transformation of human prostate cells. PLoS One 4:e6872.  https://doi.org/10.1371/journal.pone.0006872 CrossRefPubMedPubMedCentralGoogle Scholar
  45. Owen KA, Anderson CJ, Casanova JE (2016) Salmonella suppresses the TRIF-dependent type I interferon response in macrophages. MBio 7:e02051–e020515.  https://doi.org/10.1128/mBio.02051-15 CrossRefPubMedPubMedCentralGoogle Scholar
  46. Ozaki T, Nakagawara A (2011) Role of p53 in cell death and human cancers. Cancers (Basel) 3:994–1013.  https://doi.org/10.3390/cancers3010994 CrossRefPubMedCentralGoogle Scholar
  47. Pehlivan M, Itirli G, Onay H et al (2004) Does Mycoplasma sp. play role in small cell lung cancer? Lung Cancer 45:129–130.  https://doi.org/10.1016/j.lungcan.2004.01.007 CrossRefPubMedGoogle Scholar
  48. Razin S, Yogev D, Naot Y (1998) Molecular biology and pathogenicity of mycoplasmas. Microbiol Mol Biol Rev 62:1094–1156PubMedPubMedCentralGoogle Scholar
  49. Shimizu T, Kimura Y, Kida Y et al (2014) Cytadherence of Mycoplasma pneumoniae induces inflammatory responses through autophagy and toll-like receptor 4. Infect Immun 82:3076–3086.  https://doi.org/10.1128/IAI.01961-14 CrossRefPubMedPubMedCentralGoogle Scholar
  50. Tokalov SV, Abolmaali ND (2010) Protection of p53 wild type cells from taxol by nutlin-3 in the combined lung cancer treatment. BMC Cancer 10:57.  https://doi.org/10.1186/1471-2407-10-57 CrossRefPubMedPubMedCentralGoogle Scholar
  51. Vande Voorde J, Balzarini J, Liekens S (2014) Mycoplasmas and cancer: focus on nucleoside metabolism. EXCLI J 13:300–322.  https://doi.org/10.17877/DE290R-15593 PubMedPubMedCentralGoogle Scholar
  52. Wang X, Yang J, Qian J et al (2015) S100A14, a mediator of epithelial–mesenchymal transition, regulates proliferation, migration and invasion of human cervical cancer cells. Am J Cancer Res 5:1484–1495PubMedPubMedCentralGoogle Scholar
  53. Wingender E (2008) The TRANSFAC project as an example of framework technology that supports the analysis of genomic regulation. Brief Bioinform 9:326–332.  https://doi.org/10.1093/bib/bbn016 CrossRefPubMedGoogle Scholar
  54. Wrage M, Hagmann W, Kemming D et al (2015) Identification of HERC5 and its potential role in NSCLC progression. Int J Cancer 136:2264–2272.  https://doi.org/10.1002/ijc.29298 CrossRefPubMedGoogle Scholar
  55. Wu Y, Zhou BP (2008) New insights of epithelial–mesenchymal transition in cancer metastasis. Acta Biochim Biophys Sin (Shanghai) 40:643–650.  https://doi.org/10.1111/j.1745-7270.2008.00443.x CrossRefGoogle Scholar
  56. Yang J, Hooper WC, Phillips DJ, Talkington DF (2003) Interleukin-1beta responses to Mycoplasma pneumoniae infection are cell-type specific. Microb Pathog 34:17–25CrossRefPubMedGoogle Scholar
  57. Yang H, Qu L, Ma H et al (2010) Mycoplasma hyorhinis infection in gastric carcinoma and its effects on the malignant phenotypes of gastric cancer cells. BMC Gastroenterol 10:132.  https://doi.org/10.1186/1471-230X-10-132 CrossRefPubMedPubMedCentralGoogle Scholar
  58. Yokota S, Saito H, Kubota T et al (2003) Measles virus suppresses interferon-α signaling pathway: suppression of Jak1 phosphorylation and association of viral accessory proteins, C and V, with interferon-α receptor complex. Virology 306:135–146.  https://doi.org/10.1016/S0042-6822(02)00026-0 CrossRefPubMedGoogle Scholar
  59. Zhang S, Tsai S, Wu TT et al (2004) Mycoplasma fermentans infection promotes immortalization of human peripheral blood mononuclear cells in culture. Blood 104:4252–4259.  https://doi.org/10.1182/blood-2004-04-1245 CrossRefPubMedGoogle Scholar
  60. Zuo L, Wu Y, You X (2009) Mycoplasma lipoproteins and Toll-like receptors. J Zhejiang Univ Sci B 10:67–76.  https://doi.org/10.1631/jzus.B0820256 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Uljana A. Boyarskikh
    • 1
  • Alexandra S. Shadrina
    • 1
    • 2
  • Mariya A. Smetanina
    • 1
    • 2
  • Yakov A. Tsepilov
    • 2
    • 3
  • Igor P. Oscorbin
    • 1
    • 2
  • Vadim V. Kozlov
    • 4
  • Alexander E. Kel
    • 1
    • 5
  • Maxim L. Filipenko
    • 1
    • 2
  1. 1.Laboratory of PharmacogenomicsInstitute of Chemical Biology and Fundamental MedicineNovosibirskRussia
  2. 2.Novosibirsk State UniversityNovosibirskRussia
  3. 3.Institute of Cytology and GeneticsNovosibirskRussia
  4. 4.Novosibirsk Regional Clinical Oncological CenterNovosibirskRussia
  5. 5.Department of Research and DevelopmentgeneXplain GmbHWolfenbüttelGermany

Personalised recommendations