Article | Published:

Targeting surface nucleolin induces autophagy-dependent cell death in pancreatic cancer via AMPK activation

Oncogenevolume 38pages18321844 (2019) | Download Citation

Abstract

Pancreatic cancer remains one of the deadliest human cancers despite current advances in conventional therapeutics including surgery and adjuvant therapies. Here, we showed that LZ1, a peptide derived from a snake venom cathelicidin, significantly inhibited growth of pancreatic cancer cells by inducing autophagy-dependent cell death both in vitro and in vivo. The LZ1-induced cell death was blocked by pharmacological or genetic inhibition of autophagy. In orthotopic model of pancreatic cancer, systemic administration of LZ1 (1–4 mg/kg) exhibited remarkable antitumor efficacy, significantly prolonged mice survival, and showed negligible adverse effects by comparison with gemcitabine (20 mg/kg). Mechanistic studies revealed that LZ1 acts through binding to nucleolin, whose expression on cell surface is frequently increased in pancreatic cancer cells. LZ1 binding triggers degradation of surface-expressed nucleolin. This leads to activation of 5′-AMP kinase which results in suppression of mTORC1 activity and induction of autophagic flux. These data suggest that LZ1, targeting nucleolin–AMPK–autophagy axis, is a promising lead for the development of therapeutic agents against pancreatic 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.

Additional information

These authors contributed equally: Cheng Xu, Yunfei Wang, Qiu Tu, Zhiye Zhang

References

  1. 1.

    Siegel RL, Miller KD, Jemal A. Cancer statistics, 2017. CA Cancer J Clin. 2017;67:7–30.

  2. 2.

    Vincent A, Herman J, Schulick R, Hruban RH, Goggins M. Pancreatic cancer. Lancet. 2011;378:607–20.

  3. 3.

    Hidalgo M, Cascinu S, Kleeff J, Labianca R, Lohr JM, Neoptolemos J, et al. Addressing the challenges of pancreatic cancer: future directions for improving outcomes. Pancreatology. 2015;15:8–18.

  4. 4.

    de Sousa Cavalcante L, Monteiro G. Gemcitabine: metabolism and molecular mechanisms of action, sensitivity and chemoresistance in pancreatic cancer. Eur J Pharmacol. 2014;741:8–16.

  5. 5.

    Conroy T, Desseigne F, Ychou M, Bouche O, Guimbaud R, Becouarn Y, et al. FOLFIRINOX versus gemcitabine for metastatic pancreatic cancer. N Engl J Med. 2011;364:1817–25.

  6. 6.

    Shi S, Yao W, Xu J, Long J, Liu C, Yu X. Combinational therapy: new hope for pancreatic cancer? Cancer Lett. 2012;317:127–35.

  7. 7.

    Mongelard F, Bouvet P. Nucleolin: a multiFACeTed protein. Trends Cell Biol. 2007;17:80–86.

  8. 8.

    Berger CM, Gaume X, Bouvet P. The roles of nucleolin subcellular localization in cancer. Biochimie. 2015;113:78–85.

  9. 9.

    Huang Y, Shi H, Zhou H, Song X, Yuan S, Luo Y. The angiogenic function of nucleolin is mediated by vascular endothelial growth factor and nonmuscle myosin. Blood. 2006;107:3564–71.

  10. 10.

    Said EA, Courty J, Svab J, Delbe J, Krust B, Hovanessian AG. Pleiotrophin inhibits HIV infection by binding the cell surface-expressed nucleolin. FEBS J. 2005;272:4646–59.

  11. 11.

    Said EA, Krust B, Nisole S, Svab J, Briand JP, Hovanessian AG. The anti-HIV cytokine midkine binds the cell surface-expressed nucleolin as a low affinity receptor. J Biol Chem. 2002;277:37492–502.

  12. 12.

    Krust B, Vienet R, Cardona A, Rougeot C, Jacotot E, Callebaut C, et al. The anti-HIV pentameric pseudopeptide HB-19 is preferentially taken up in vivo by lymphoid organs where it forms a complex with nucleolin. Proc Natl Acad Sci USA. 2001;98:14090–5.

  13. 13.

    Soundararajan S, Chen W, Spicer EK, Courtenay-Luck N, Fernandes DJ. The nucleolin targeting aptamer AS1411 destabilizes Bcl-2 messenger RNA in human breast cancer cells. Cancer Res. 2008;68:2358–65.

  14. 14.

    Reyes-Reyes EM, Teng Y, Bates PJ. A new paradigm for aptamer therapeutic AS1411 action: uptake by macropinocytosis and its stimulation by a nucleolin-dependent mechanism. Cancer Res. 2010;70:8617–29.

  15. 15.

    Krust B, El Khoury D, Nondier I, Soundaramourty C, Hovanessian AG. Targeting surface nucleolin with multivalent HB-19 and related Nucant pseudopeptides results in distinct inhibitory mechanisms depending on the malignant tumor cell type. BMC Cancer. 2011;11:333.

  16. 16.

    Benedetti E, Antonosante A, d’Angelo M, Cristiano L, Galzio R, Destouches D, et al. Nucleolin antagonist triggers autophagic cell death in human glioblastoma primary cells and decreased in vivo tumor growth in orthotopic brain tumor model. Oncotarget. 2015;6:42091–104.

  17. 17.

    Palmieri D, Richmond T, Piovan C, Sheetz T, Zanesi N, Troise F, et al. Human anti-nucleolin recombinant immunoagent for cancer therapy. Proc Natl Acad Sci USA. 2015;112:9418–23.

  18. 18.

    Gilles ME, Maione F, Cossutta M, Carpentier G, Caruana L, Di Maria S, et al. Nucleolin targeting impairs the progression of pancreatic cancer and promotes the normalization of tumor vasculature. Cancer Res. 2016;76:7181–93.

  19. 19.

    Wu WK, Wang G, Coffelt SB, Betancourt AM, Lee CW, Fan D, et al. Emerging roles of the host defense peptide LL-37 in human cancer and its potential therapeutic applications. Int J Cancer. 2010;127:1741–7.

  20. 20.

    Ren SX, Cheng AS, To KF, Tong JH, Li MS, Shen J, et al. Host immune defense peptide LL-37 activates caspase-independent apoptosis and suppresses colon cancer. Cancer Res. 2012;72:6512–23.

  21. 21.

    Wang H, Ke M, Tian Y, Wang J, Li B, Wang Y, et al. BF-30 selectively inhibits melanoma cell proliferation via cytoplasmic membrane permeabilization and DNA-binding in vitro and in B16F10-bearing mice. Eur J Pharmacol. 2013;707:1–10.

  22. 22.

    Nikoletopoulou V, Markaki M, Palikaras K, Tavernarakis N. Crosstalk between apoptosis, necrosis and autophagy. Biochim Biophys Acta. 2013;1833:3448–59.

  23. 23.

    Paglin S, Hollister T, Delohery T, Hackett N, McMahill M, Sphicas E, et al. A novel response of cancer cells to radiation involves autophagy and formation of acidic vesicles. Cancer Res. 2001;61:439–44.

  24. 24.

    Mizushima N, Yoshimori T, Levine B. Methods in mammalian autophagy research. Cell. 2010;140:313–26.

  25. 25.

    Komatsu M, Waguri S, Koike M, Sou YS, Ueno T, Hara T, et al. Homeostatic levels of p62 control cytoplasmic inclusion body formation in autophagy-deficient mice. Cell. 2007;131:1149–63.

  26. 26.

    Seglen PO, Gordon PB. 3-Methyladenine: specific inhibitor of autophagic/lysosomal protein degradation in isolated rat hepatocytes. Proc Natl Acad Sci USA. 1982;79:1889–92.

  27. 27.

    He C, Klionsky DJ. Regulation mechanisms and signaling pathways of autophagy. Annu Rev Genet. 2009;43:67–93.

  28. 28.

    Mihaylova MM, Shaw RJ. The AMPK signalling pathway coordinates cell growth, autophagy and metabolism. Nat Cell Biol. 2011;13:1016–23.

  29. 29.

    Hovanessian AG. Midkine, a cytokine that inhibits HIV infection by binding to the cell surface expressed nucleolin. Cell Res. 2006;16:174–81.

  30. 30.

    Nazli O, Bozdag AD, Tansug T, Kir R, Kaymak E. The diagnostic importance of CEA and CA 19-9 for the early diagnosis of pancreatic carcinoma. Hepatogastroenterology. 2000;47:1750–2.

  31. 31.

    Kim MP, Gallick GE. Gemcitabine resistance in pancreatic cancer: picking the key players. Clin Cancer Res. 2008;14:1284–5.

  32. 32.

    Ryan DP, Hong TS, Bardeesy N. Pancreatic adenocarcinoma. N Engl J Med. 2014;371:1039–49.

  33. 33.

    Kroemer G, Levine B. Autophagic cell death: the story of a misnomer. Nat Rev Mol Cell Biol. 2008;9:1004–10.

  34. 34.

    Shen S, Kepp O, Kroemer G. The end of autophagic cell death? Autophagy. 2012;8:1–3.

  35. 35.

    Yang A, Herter-Sprie G, Zhang H, Lin EY, Biancur D, Wang X. et al. Autophagy sustains pancreatic cancer growth through both cell autonomous and non-autonomous mechanisms. Cancer Discov. 2018;8:276–87.

  36. 36.

    Yang S, Wang X, Contino G, Liesa M, Sahin E, Ying H, et al. Pancreatic cancers require autophagy for tumor growth. Genes Dev. 2011;25:717–29.

  37. 37.

    Liu Y, Levine B. Autosis and autophagic cell death: the dark side of autophagy. Cell Death Differ. 2015;22:367–76.

  38. 38.

    Zhou S, Zhao L, Kuang M, Zhang B, Liang Z, Yi T, et al. Autophagy in tumorigenesis and cancer therapy: Dr. Jekyll or Mr. Hyde? Cancer Lett. 2012;323:115–27.

  39. 39.

    Hamma-Kourbali Y, Bermek O, Bernard-Pierrot I, Karaky R, Martel-Renoir D, Frechault S, et al. The synthetic peptide P111-136 derived from the C-terminal domain of heparin affin regulatory peptide inhibits tumour growth of prostate cancer PC-3 cells. BMC Cancer. 2011;11:212.

  40. 40.

    Galzio R, Rosati F, Benedetti E, Cristiano L, Aldi S, Mei S, et al. Glycosilated nucleolin as marker for human gliomas. J Cell Biochem. 2012;113:571–9.

  41. 41.

    Li F, Lu J, Liu J, Liang C, Wang M, Wang L, et al. A water-soluble nucleolin aptamer-paclitaxel conjugate for tumor-specific targeting in ovarian cancer. Nat Commun. 2017;8:1390.

  42. 42.

    Destouches D, Page N, Hamma-Kourbali Y, Machi V, Chaloin O, Frechault S, et al. A simple approach to cancer therapy afforded by multivalent pseudopeptides that target cell-surface nucleoproteins. Cancer Res. 2011;71:3296–305.

  43. 43.

    Chen CM, Chiang SY, Yeh NH. Increased stability of nucleolin in proliferating cells by inhibition of its self-cleaving activity. J Biol Chem. 1991;266:7754–8.

  44. 44.

    Fang SH, Yeh NH. The self-cleaving activity of nucleolin determines its molecular dynamics in relation to cell proliferation. Exp Cell Res. 1993;208:48–53.

  45. 45.

    Dhanabal M, Ramchandran R, Waterman MJ, Lu H, Knebelmann B, Segal M, et al. Endostatin induces endothelial cell apoptosis. J Biol Chem. 1999;274:11721–6.

  46. 46.

    Farin K, Schokoroy S, Haklai R, Cohen-Or I, Elad-Sfadia G, Reyes-Reyes ME, et al. Oncogenic synergism between ErbB1, nucleolin, and mutant Ras. Cancer Res. 2011;71:2140–51.

  47. 47.

    Goldshmit Y, Trangle SS, Kloog Y, Pinkas-Kramarski R. Interfering with the interaction between ErbB1, nucleolin and Ras as a potential treatment for glioblastoma. Oncotarget. 2014;5:8602–13.

  48. 48.

    He X, Shen C, Lu Q, Li J, Wei Y, He L, et al. Prokineticin 2 plays a pivotal role in psoriasis. EBioMedicine. 2016;13:248–61.

  49. 49.

    Khanbolooki S, Nawrocki ST, Arumugam T, Andtbacka R, Pino MS, Kurzrock R, et al. Nuclear factor-kappaB maintains TRAIL resistance in human pancreatic cancer cells. Mol Cancer Ther. 2006;5:2251–60.

  50. 50.

    Qin J, Zhou Z, Chen W, Wang C, Zhang H, Ge G, et al. BAP1 promotes breast cancer cell proliferation and metastasis by deubiquitinating KLF5. Nat Commun. 2015;6:8471.

Download references

Acknowledgements

This work was supported by National Natural Science Foundation of China (21761142002 and 81770464), Ministry of Science and Technology (2018ZX09301043-003), Chinese Academy of Science (QYZDJ-SSWSMC012, SAJC201606, the West Light Foundation and Youth Innovation Promotion Association (2017432)) and Yunnan Provincial Science and Technology Department (2017FB038, 2015BC005).

Author information

Author notes

    Affiliations

    1. Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences/Key Laboratory of Bioactive Peptides of Yunnan Province, Kunming Institute of Zoology, Kunming, 650223, Yunnan, China

      • Cheng Xu
      • , Yunfei Wang
      • , Qiu Tu
      • , Zhiye Zhang
      • , James Mwangi
      • , Xudong Zhao
      •  & Ren Lai
    2. Kunming College of Life Science, University of Chinese Academy of Sciences, Kunming, 650204, Yunnan, China

      • Cheng Xu
      • , Yunfei Wang
      •  & James Mwangi
    3. Life Sciences College of Nanjing Agricultural University, Nanjing, 210095, Jiangsu, China

      • Mengrou Chen
      •  & Ren Lai
    4. Sino-African Joint Research Center, Chinese Academy of Sciences, Kunming Institute of Zoology, Kunming, 650223, Yunnan, China

      • James Mwangi
      •  & Ren Lai
    5. Department of Cardiovascular Surgery, Yan’an Affiliated Hospital of Kunming Medical University, Kunming, 650041, Yunnan, China

      • Yaxiong Li
    6. Institute for Cancer Genetics and Informatics, Oslo University Hospital, Oslo, Norway

      • Yang Jin

    Authors

    1. Search for Cheng Xu in:

    2. Search for Yunfei Wang in:

    3. Search for Qiu Tu in:

    4. Search for Zhiye Zhang in:

    5. Search for Mengrou Chen in:

    6. Search for James Mwangi in:

    7. Search for Yaxiong Li in:

    8. Search for Yang Jin in:

    9. Search for Xudong Zhao in:

    10. Search for Ren Lai in:

    Conflict of interest

    The authors declare that they have no conflict of interest.

    Corresponding authors

    Correspondence to Yang Jin or Xudong Zhao or Ren Lai.

    Electronic supplementary material

    About this article

    Publication history

    Received

    Revised

    Accepted

    Published

    Issue Date

    DOI

    https://doi.org/10.1038/s41388-018-0556-x