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Extracellular HSP70-peptide complexes promote the proliferation of hepatocellular carcinoma cells via TLR2/4/JNK1/2MAPK pathway

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Tumor Biology

Abstract

Heat shock protein 70 (HSP70) and HSP70-peptide complexes (HSP70-PCs) have been implicated in the pathogenesis of multiple tumors in humans and have been experimentally shown to increase the proliferation of cell lines derived from hepatocellular carcinoma. The goal of this study was to elucidate the molecular mechanisms through which extracellular HSP70/HSP70-PCs stimulate the proliferation of hepatocellular carcinoma (HCC). The molecular mechanisms of HSP70/HSP70-PC action were studied in the human hepatocellular carcinoma cell lines HepG2 and Huh-7, as well as tumor tissue collected from patients with HCC (n = 95). We found that HSP70/HSP70-PCs can stimulate the proliferation of HepG2 cells and that this effect is blocked by knocking down TLR2 and TLR4 expression by RNA interference. A physical interaction between HSP70/HSP70-PCs and TLR2/4 was established using co-immunoprecipitation and pull-down assays. Pharmacological inhibition of different branches of the MAPK intracellular signaling pathway indicated that the extracellular HSP70/HSP70-PC effect was mediated by the JNK1/2 signaling pathway within the cell. We also studied TLR2 and TLR expression at the protein and messenger RNA (mRNA) level in tumor and non-tumor tissue in patients with HCC (n = 95), finding that TLR2 and 4 are increased in HCC tumor tissue and that the expression of TLR2 correlates with clinicopathologic features of HCC. Our data conclusively demonstrates that extracellular HSP70/HSP70-PCs can promote the proliferation of HCC cells through activation of TLR2 and TLR4 and subsequent activation of the intracellular JNK1/2/MAPK signaling pathway.

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References

  1. Torre LA et al. Global cancer statistics, 2012. CA Cancer J Clin. 2015;65(2):87–108.

    Article  PubMed  Google Scholar 

  2. Chen JG, Zhang SW. Liver cancer epidemic in China: past, present and future. Semin Cancer Biol. 2011;21(1):59–69.

    Article  PubMed  Google Scholar 

  3. Maeda S. NF-kappaB, JNK, and TLR signaling pathways in hepatocarcinogenesis. Gastroenterol Res Pract. 2010;2010:367694.

    Article  PubMed  PubMed Central  Google Scholar 

  4. Duffy DJ, Millane RC, Frank U. A heat shock protein and Wnt signaling crosstalk during axial patterning and stem cell proliferation. Dev Biol. 2012;362(2):271–81.

    Article  CAS  PubMed  Google Scholar 

  5. Kang Y et al. Prognostic significance of heat shock protein 70 expression in early gastric carcinoma. Korean J Pathol. 2013;47(3):219–26.

    Article  PubMed  PubMed Central  Google Scholar 

  6. Gogate SS et al. Tonicity enhancer binding protein (TonEBP) and hypoxia-inducible factor (HIF) coordinate heat shock protein 70 (Hsp70) expression in hypoxic nucleus pulposus cells: role of Hsp70 in HIF-1alpha degradation. J Bone Miner Res. 2012;27(5):1106–17.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Horibe T et al. Synergetic cytotoxic activity toward breast cancer cells enhanced by the combination of Antp-TPR hybrid peptide targeting Hsp90 and Hsp70-targeted peptide. BMC Cancer. 2014;14:615.

    Article  PubMed  PubMed Central  Google Scholar 

  8. Schmitt E et al. Intracellular and extracellular functions of heat shock proteins: repercussions in cancer therapy. J Leukoc Biol. 2007;81(1):15–27.

    Article  CAS  PubMed  Google Scholar 

  9. Wu FH et al. Extracellular HSPA1A promotes the growth of hepatocarcinoma by augmenting tumor cell proliferation and apoptosis-resistance. Cancer Lett. 2012;317(2):157–64.

    Article  CAS  PubMed  Google Scholar 

  10. Asea A et al. Novel signal transduction pathway utilized by extracellular HSP70: role of toll-like receptor (TLR) 2 and TLR4. J Biol Chem. 2002;277(17):15028–34.

    Article  CAS  PubMed  Google Scholar 

  11. Murphy MB, Medvedev AE. Long noncoding RNAs as regulators of Toll-like receptor signaling and innate immunity. J Leukoc Biol. 2016.

  12. Chen L, Yu J. Modulation of Toll-like receptor signaling in innate immunity by natural products. Int Immunopharmacol. 2016.

  13. Tsai SY et al. DAMP molecule S100 A9 acts as a molecular pattern to enhance inflammation during influenza a virus infection: role of DDX21-TRIF-TLR4-MyD88 pathway. PLoS Pathog. 2014;10(1):e1003848.

    Article  PubMed  PubMed Central  Google Scholar 

  14. Henrick BM et al. HIV-1 structural proteins serve as PAMPs for TLR2 heterodimers significantly increasing infection and innate immune activation. Front Immunol. 2015;6:426.

    PubMed  PubMed Central  Google Scholar 

  15. Leake I. Hepatocellular carcinoma. Treatment potential of targeting Toll-like receptors in HCC. Nat Rev. Gastroenterol Hepatol. 2014;11(9):518.

    Google Scholar 

  16. Chen R et al. Cancers take their Toll—the function and regulation of toll-like receptors in cancer cells. Oncogene. 2008;27(2):225–33.

    Article  CAS  PubMed  Google Scholar 

  17. Yue P et al. Des-gamma-carboxyl prothrombin induces matrix metalloproteinase activity in hepatocellular carcinoma cells by involving the ERK1/2 MAPK signalling pathway. Eur J Cancer. 2011;47(7):1115–24.

    Article  CAS  PubMed  Google Scholar 

  18. Huang Y, et al. MAPK/ERK2 phosphorylates ERG at serine 283 in leukemic cells and promotes stem cell signatures and cell proliferation. Leukemia. 2016.

  19. Kamiyama M, Naguro I, Ichijo H. In vivo gene manipulation reveals the impact of stress-responsive MAPK pathways on tumor progression. Cancer Sci. 2015;106(7):785–96.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Peroval MY et al. A critical role for MAPK signalling pathways in the transcriptional regulation of toll like receptors. PLoS One. 2013;8(2):e51243.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Wu TT et al. GATA-2 transduces LPS-induced il-1beta gene expression in macrophages via a toll-like receptor 4/MD88/MAPK-dependent mechanism. PLoS One. 2013;8(8):e72404.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Shimura T, Fukumoto M, Kunugita N. The role of cyclin D1 in response to long-term exposure to ionizing radiation. Cell Cycle. 2013;12(17):2738–43.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Zhang Y et al. A systematic investigation based on microRNA-mediated gene regulatory network reveals that dysregulation of microRNA-19a/cyclin D1 axis confers an oncogenic potential and a worse prognosis in human hepatocellular carcinoma. RNA Biol. 2015;12(6):643–57.

    Article  PubMed  PubMed Central  Google Scholar 

  24. Qiu C et al. GM-CSF induces cyclin D1 expression and proliferation of endothelial progenitor cells via PI3K and MAPK signaling. Cell Physiol Biochem. 2014;33(3):784–95.

    Article  CAS  PubMed  Google Scholar 

  25. Gao Y et al. A new purification method for enhancing the immunogenicity of heat shock protein 70-peptide complexes. Oncol Rep. 2012;28(6):1977–83.

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Li H et al. Expression of HSP70 and JNK-related proteins in human liver cancer: potential effects on clinical outcome. Dig Liver Dis. 2007;39(7):663–70.

    Article  CAS  PubMed  Google Scholar 

  27. Blachere NE et al. Heat shock protein-peptide complexes, reconstituted in vitro, elicit peptide-specific cytotoxic T lymphocyte response and tumor immunity. J Exp Med. 1997;186(8):1315–22.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Casaburi I et al. Chenodeoxycholic acid through a TGR5-dependent CREB signaling activation enhances cyclin D1 expression and promotes human endometrial cancer cell proliferation. Cell Cycle. 2012;11(14):2699–710.

    Article  CAS  PubMed  Google Scholar 

  29. Kiang JG, Tsokos GC. Heat shock protein 70 kDa: molecular biology, biochemistry, and physiology. Pharmacol Ther. 1998;80(2):183–201.

    Article  CAS  PubMed  Google Scholar 

  30. Cho HS et al. Enhanced HSP70 lysine methylation promotes proliferation of cancer cells through activation of Aurora kinase B. Nat Commun. 2012;3:1072.

    Article  PubMed  PubMed Central  Google Scholar 

  31. Gong W et al. Invasion potential of H22 hepatocarcinoma cells is increased by HMGB1-induced tumor NF-kappaB signaling via initiation of HSP70. Oncol Rep. 2013;30(3):1249–56.

    CAS  PubMed  Google Scholar 

  32. Monma H et al. The HSP70 and autophagy inhibitor pifithrin-mu enhances the antitumor effects of TRAIL on human pancreatic cancer. Mol Cancer Ther. 2013;12(4):341–51.

    Article  CAS  PubMed  Google Scholar 

  33. Mambula SS, Calderwood SK. Heat shock protein 70 is secreted from tumor cells by a nonclassical pathway involving lysosomal endosomes. J Immunol. 2006;177(11):7849–57.

    Article  CAS  PubMed  Google Scholar 

  34. Bohnhorst J et al. Toll-like receptors mediate proliferation and survival of multiple myeloma cells. Leukemia. 2006;20(6):1138–44.

    Article  CAS  PubMed  Google Scholar 

  35. Gandhi A, Moorthy B, Ghose R. Drug disposition in pathophysiological conditions. Curr Drug Metab. 2012;13(9):1327–44.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Lee CH, Wu CL, Shiau AL. Toll-like receptor 4 signaling promotes tumor growth. J Immunother. 2010;33(1):73–82.

    Article  CAS  PubMed  Google Scholar 

  37. Yu L, Wang L, Chen S. Dual character of toll-like receptor signaling: pro-tumorigenic effects and anti-tumor functions. Biochim Biophys Acta. 2013;1835(2):144–54.

    CAS  PubMed  Google Scholar 

  38. Jain RK. Normalizing tumor microenvironment to treat cancer: bench to bedside to biomarkers. J Clin Oncol. 2013;31(17):2205–18.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Kim JY et al. Sulforaphane suppresses vascular adhesion molecule-1 expression in TNF-alpha-stimulated mouse vascular smooth muscle cells: involvement of the MAPK, NF-kappaB and AP-1 signaling pathways. Vasc Pharmacol. 2012;56(3–4):131–41.

    Article  CAS  Google Scholar 

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Acknowledgments

This study is supported by the National Natural Science Foundation of China (No. 81071955) and the Scientific Research from Educational Department of Liaoning Province, China (No. 2013021088).

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Authors and Affiliations

  1. School of Stomatology, China Medical University, Shenyang, China

    Yi Zhe

  2. Department of Oncology, Tumour Angiogenesis and Microenvironment Laboratory (TAML), First Affiliated Hospital, Liaoning Medical College, Jinzhou, China

    Yan Li

  3. Department of General Surgery, The Fourth Affiliated Hospital of China Medical University, Shenyang, 110004, Liaoning Province, China

    Dan Liu, Jin-Gang Liu & Hang-Yu Li

  4. Center of Metabolic Disease Research, Nanjing Medical University, Nanjing, China

    Dong-Ming Su

Authors
  1. Yi Zhe
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  2. Yan Li
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  3. Dan Liu
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  5. Jin-Gang Liu
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  6. Hang-Yu Li
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Corresponding author

Correspondence to Hang-Yu Li.

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The Institutional Review Board of the Shengjing Hospital of China Medical University approved the use of human tissue samples for this project. All patients gave their informed and written consent for the use of the clinical specimens for research.

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Zhe, Y., Li, Y., Liu, D. et al. Extracellular HSP70-peptide complexes promote the proliferation of hepatocellular carcinoma cells via TLR2/4/JNK1/2MAPK pathway. Tumor Biol. 37, 13951–13959 (2016). https://doi.org/10.1007/s13277-016-5189-5

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  • DOI: https://doi.org/10.1007/s13277-016-5189-5

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