Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Non-toxic fragment of botulinum neurotoxin type A and monomethyl auristatin E conjugate for targeted therapy for neuroendocrine tumors

Cancer Gene Therapy volume 27, pages 898–909 (2020)Cite this article

Subjects

Abstract

Surgical resection is the only cure for neuroendocrine tumors (NETs). However, widespread metastases have already occured by the time of initial diagnosis in many cases making complete surgical removal impossible. We developed a recombinant heavy-chain receptor binding domain (rHCR) of botulinum neurotoxin type A that can specifically target synaptic vesicle 2 (SV2), a surface receptor abundantly expressed in multiple neuroendocrine tumors. Expression of neuroendocrine differentiation markers chromogranin A (CgA) and achaete-scute complex 1 (ASCL1) were signficantly reduced when treated with rHCR. rHCR conjugated to the antimitotic agent monomethyl auristatin E (MMAE) significantly suppressed proliferation of pancreatic carcinoid (BON) and medullary thyroid cancer cells (MZ) at concentrations of 500 and 300 nM respectively, while no growth suppression was observed in pulmonary fibroblasts and cortical neuron control cell lines. In vivo, rHCR-MMAE significantly reduced tumor volume in mouse xenografts with no observed adverse effects. These data suggest recombinant HCR (rHCR) of BoNT/A preferentially targets neuroendocrine cancer without the neurotoxicity of the full BoNT/A and that SV2 is a specific and promising target for delivering drugs to neuroendocrine tumors.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Synaptic vesicle protein expression and rHCR uptake.
Fig. 2: Western blots and quantitative analyses of SV-mediated SNAP25 cleavage of all SNAP25 expressing cell lines treated with 5 ng/μl (100 nM) of BoNT/A and analyzed for SNAP25 cleavage by Western blot.
Fig. 3: Suppression of CgA and ASCL1 in rHCR treated NE cancer cell lines.
Fig. 4: Monomethylaurostatin E cytotoxicity in neuroendocrine tumor cells.
Fig. 5: Comparative cytotoxicity of rHCR and rHCR-MMAE conjugate.
Fig. 6: In vivo anticancer efficacy of rHCR-MMAE in BON tumor xenografts.

Similar content being viewed by others

References

  1. Barakat MT, Meeran K, Bloom SR. Neuroendocrine tumours. Endocr Relat Cancer. 2004;11:1–18.

    CAS  PubMed  Google Scholar 

  2. Chen CQ, Chen HL, Cai SR, Wang Z, Ma JP, Zhang CH, et al. [Clinicopathologic features, diagnosis and treatment of 38 neuroendocrine carcinoma in the digestive system]. Zhonghua Wei Chang Wai Ke Za Zhi. 2010;13:587–9.

    PubMed  Google Scholar 

  3. Pinchot SN, Holen K, Sippel RS, Chen H. Carcinoid tumors. Oncologist. 2008;13:1255–69.

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Massironi S, Rossi RE, Casazza G, Conte D, Ciafardini C, Galeazzi M, et al. Chromogranin A in diagnosing and monitoring patients with gastroenteropancreatic neuroendocrine neoplasms: a large series from a single institution. Neuroendocrinology. 2014;100:240–9.

    CAS  PubMed  Google Scholar 

  5. Yao JC, Hassan M, Phan A, Dagohoy C, Leary C, Mares JE, et al. One hundred years after “carcinoid”: epidemiology of and prognostic factors for neuroendocrine tumors in 35,825 cases in the United States. J Clin Oncol. 2008;26:3063–72.

    PubMed  Google Scholar 

  6. Taal BG, Visser O. Epidemiology of neuroendocrine tumours. Neuroendocrinology. 2004;80(Suppl 1):3–7.

    CAS  PubMed  Google Scholar 

  7. Metz DC, Jensen RT. Gastrointestinal neuroendocrine tumors: pancreatic endocrine tumors. Gastroenterology. 2008;135:1469–92.

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Demirkan BH, Eriksson B. Systemic treatment of neuroendocrine tumors with hepatic metastases. Turk J Gastroenterol. 2012;23:427–37.

    PubMed  Google Scholar 

  9. Barbieri F, Albertelli M, Grillo F, Mohamed A, Saveanu A, Barlier A, et al. Neuroendocrine tumors: insights into innovative therapeutic options and rational development of targeted therapies. Drug Discov Today. 2014;19:458–68.

    CAS  PubMed  Google Scholar 

  10. Gulenchyn KY, Yao X, Asa SL, Singh S, Law C. Radionuclide therapy in neuroendocrine tumours: a systematic review. Clin Oncol (R Coll Radiol). 2012;24:294–308.

    CAS  Google Scholar 

  11. Righi L, Volante M, Tavaglione V, Bille A, Daniele L, Angusti T, et al. Somatostatin receptor tissue distribution in lung neuroendocrine tumours: a clinicopathologic and immunohistochemical study of 218 ‘clinically aggressive’ cases. Ann Oncol. 2010;21:548–55.

    CAS  PubMed  Google Scholar 

  12. Kwekkeboom DJ, de Herder WW, Kam BL, van Eijck CH, van Essen M, Kooij PP. et al. Treatment with the radiolabeled somatostatin analog [177 Lu-DOTA 0,Tyr3]octreotate: toxicity, efficacy, and survival. J Clin Oncol. 2008;26:2124–30.

    CAS  Google Scholar 

  13. Barbieri JS, Seshasai R, Shemesh A, Sedrak M, Hoffman B, Alley EW. Thymic neuroendocrine tumor presenting with the ectopic ACTH syndrome. J Thorac Oncol. 2013;8:e57–8.

    PubMed  Google Scholar 

  14. Grillo F, Florio T, Ferrau F, Kara E, Fanciulli G, Faggiano A, et al. Emerging multitarget tyrosine kinase inhibitors in the treatment of neuroendocrine neoplasms. Endocr Relat Cancer. 2018;25:R453–66.

    PubMed  Google Scholar 

  15. Yao JC, Shah MH, Ito T, Bohas CL, Wolin EM, Van Cutsem E, et al. Everolimus for advanced pancreatic neuroendocrine tumors. N Engl J Med. 2011;364:514–23.

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Pool SE, Bison S, Koelewijn SJ, van der Graaf LM, Melis M, Krenning EP, et al. mTOR inhibitor RAD001 promotes metastasis in a rat model of pancreatic neuroendocrine cancer. Cancer Res. 2013;73:12–8.

    CAS  PubMed  Google Scholar 

  17. Mohammed TA, Holen KD, Jaskula-Sztul R, Mulkerin D, Lubner SJ, Schelman WR, et al. A pilot phase II study of valproic acid for treatment of low-grade neuroendocrine carcinoma. Oncologist. 2011;16:835–43.

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Dasari A, Phan A, Gupta S, Rashid A, Yeung SC, Hess K, et al. Phase I study of the anti-IGF1R antibody cixutumumab with everolimus and octreotide in advanced well-differentiated neuroendocrine tumors. Endocr Relat Cancer. 2015;22:431–41.

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Portela-Gomes GM, Lukinius A, Grimelius L. Synaptic vesicle protein 2, A new neuroendocrine cell marker. Am J Pathol. 2000;157:1299–309.

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Jakobsen AM, Ahlman H, Wangberg B, Kolby L, Bengtsson M, Nilsson O. Expression of synaptic vesicle protein 2 (SV2) in neuroendocrine tumours of the gastrointestinal tract and pancreas. J Pathol. 2002;196:44–50.

    CAS  PubMed  Google Scholar 

  21. Nilsson O, Jakobsen AM, Kolby L, Bernhardt P, Forssell-Aronsson E, Ahlman H. Importance of vesicle proteins in the diagnosis and treatment of neuroendocrine tumors. Ann N Y Acad Sci. 2004;1014:280–3.

    CAS  PubMed  Google Scholar 

  22. Chang WP, Sudhof TC. SV2 renders primed synaptic vesicles competent for Ca2 + -induced exocytosis. J Neurosci. 2009;29:883–97.

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Hou JC, Min L, Pessin JE. Insulin granule biogenesis, trafficking and exocytosis. Vitam Horm. 2009;80:473–506.

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Keller JE, Cai F, Neale EA. Uptake of botulinum neurotoxin into cultured neurons. Biochemistry. 2004;43:526–32.

    CAS  PubMed  Google Scholar 

  25. Masuyer G, Chaddock JA, Foster KA, Acharya KR. Engineered botulinum neurotoxins as new therapeutics. Annu Rev Pharmacol Toxicol. 2014;54:27–51.

    CAS  PubMed  Google Scholar 

  26. Montal M. Botulinum neurotoxin: a marvel of protein design. Annu Rev Biochem. 2010;79:591–617.

    CAS  PubMed  Google Scholar 

  27. Moskowitz CH, Walewski J, Nademanee A, Masszi T, Agura E, Holowiecki J, et al. Five-year PFS from the AETHERA trial of brentuximab vedotin for Hodgkin lymphoma at high risk of progression or relapse. Blood. 2018;132:2639–42.

    CAS  PubMed  Google Scholar 

  28. Almhanna K, Wright D, Mercade TM, Van Laethem JL, Gracian AC, Guillen-Ponce C, et al. A phase II study of antibody-drug conjugate, TAK-264 (MLN0264) in previously treated patients with advanced or metastatic pancreatic adenocarcinoma expressing guanylyl cyclase C. Invest New Drugs. 2017;35:634–41.

    CAS  PubMed  Google Scholar 

  29. Ott PA, Hamid O, Pavlick AC, Kluger H, Kim KB, Boasberg PD, et al. Phase I/II study of the antibody-drug conjugate glembatumumab vedotin in patients with advanced melanoma. J Clin Oncol. 2014;32:3659–66.

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Arsenault J, Ferrari E, Niranjan D, Cuijpers SA, Gu C, Vallis Y, et al. Stapling of the botulinum type A protease to growth factors and neuropeptides allows selective targeting of neuroendocrine cells. J Neurochem. 2013;126:223–33.

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Xu N, Ma C, Ou J, Sun WW, Zhou L, Hu H, et al. Comparative Proteomic Analysis of Three Chinese Hamster Ovary (CHO) Host Cells. Biochem Eng J. 2017;124:122–9.

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Xu N, Ou J, Gilani A-K, Zhou L, Liu M. High-level expression of recombinant IgG1 by CHO K1 platform. Frontiers of Chemical Science and Engineering. 2015;9:376–80.

  33. Polakis P. Antibody drug conjugates for cancer therapy. Pharmacol Rev. 2016;68:3–19.

    CAS  PubMed  Google Scholar 

  34. Zarebczan B, Pinchot SN, Kunnimalaiyaan M, Chen H. Hesperetin, a potential therapy for carcinoid cancer. Am J Surg. 2011;201:329–32.

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Khan A, Gillis K, Clor J, Tyagarajan K. Simplified evaluation of apoptosis using the Muse cell analyzer. Postepy Biochem. 2012;58:492–6.

    CAS  PubMed  Google Scholar 

  36. Conover WJ. Practical Nonparametric Statistics. 3rd ed. Hoboken, NJ: Wiley; 1999.

    Google Scholar 

  37. Tilkian AG, Conover MB. Understanding heart sounds and murmurs. xii. Philadelphia: Saunders; 1979. p. 122.

    Google Scholar 

  38. Holm S. A simple sequentially rejective multiple test procedure. Scand J Stat. 1979;6:65–70.

    Google Scholar 

  39. Dong M, Yeh F, Tepp WH, Dean C, Johnson EA, Janz R, et al. SV2 is the protein receptor for botulinum neurotoxin A. Science. 2006;312:592–6.

    CAS  PubMed  Google Scholar 

  40. Hong WS, Young EW, Tepp WH, Johnson EA, Beebe DJ. A microscale neuron and Schwann cell coculture model for increasing detection sensitivity of botulinum neurotoxin type A. Toxicol Sci. 2013;134:64–72.

    CAS  PubMed  Google Scholar 

  41. Martins RG, Rajendran JG, Capell P, Byrd DR, Mankoff DA. Medullary thyroid cancer: options for systemic therapy of metastatic disease? J Clin Oncol. 2006;24:1653–5.

    PubMed  Google Scholar 

  42. Chang CL, Munin MC, Skidmore ER, Niyonkuru C, Huber LM, Weber DJ. Effect of baseline spastic hemiparesis on recovery of upper-limb function following botulinum toxin type A injections and postinjection therapy. Arch Phys Med Rehabil. 2009;90:1462–8.

    PubMed  PubMed Central  Google Scholar 

  43. Nowack A, Yao J, Custer KL, Bajjalieh SM. SV2 regulates neurotransmitter release via multiple mechanisms. Am J Physiol Cell Physiol. 2010;299:C960–7.

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Hong WS, Pezzi HM, Schuster AR, Berry SM, Sung KE, Beebe DJ. Development of a highly sensitive cell-based assay for detecting botulinum neurotoxin type A through neural culture media optimization. J Biomol Screen. 2016;21:65–73.

    CAS  PubMed  Google Scholar 

  45. Moskowitz CH, Walewski J, Nademanee A, Masszi T, Agura E, Holowiecki J, et al. Five-year PFS from the AETHERA trial of brentuximab vedotin for Hodgkin lymphoma at high risk of progression or relapse. Blood. 2018;132:2639–42.

  46. Bendell J, Saleh M, Rose AA, Siegel PM, Hart L, Sirpal S, et al. Phase I/II study of the antibody-drug conjugate glembatumumab vedotin in patients with locally advanced or metastatic breast cancer. J Clin Oncol. 2014;32:3619–25.

    CAS  PubMed  Google Scholar 

  47. Simpson LL, Rapport MM. The binding of botulinum toxin to membrane lipids: sphingolipids, steroids and fatty acids. J Neurochem. 1971;18:1751–9.

    CAS  PubMed  Google Scholar 

  48. Yowler BC, Kensinger RD, Schengrund CL. Botulinum neurotoxin A activity is dependent upon the presence of specific gangliosides in neuroblastoma cells expressing synaptotagmin I. J Biol Chem. 2002;277:32815–9.

    CAS  PubMed  Google Scholar 

  49. Dong M, Liu H, Tepp WH, Johnson EA, Janz R, Chapman ER. Glycosylated SV2A and SV2B mediate the entry of botulinum neurotoxin E into neurons. Mol Biol Cell. 2008;19:5226–37.

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank Dr. Eric Johnson, Sabine Pellett and William Tepp at the University of Wisconsin-Madison and Dr. Joseph Barbieri at the Medical College of Wisconsin, Milwaukee, WI for supplying BoNT/A and rHCR for this study. We also thank Drs. Margaret X. Liu and Jianfa Ou from University of Alabama at Birmingham for rHCR-MMAE conjugate.

Funding

This research was supported by the National Research Service Award (NRSA) T32 EB011434 and CCTS Partner Network Multidisciplinary Pilot Program award at UAB.

Author information

Author notes

  1. These authors contributed equally: Jason Whitt, Won S. Hong

Authors and Affiliations

  1. Department of Surgery, University of Alabama at Birmingham, Birmingham, AL, USA

    Jason Whitt, Rahul R. Telange, James Bibb, Herbert Chen & Renata Jaskula-Sztul

  2. Pathology and Laboratory Medicine and Biomedical Engineering, UW-Madison, Madison, WI, USA

    Won S. Hong & David J. Beebe

  3. Center for Clinical and Translational Science, University of Alabama at Birmingham, Birmingham, AL, USA

    Chee Paul Lin

Authors
  1. Jason Whitt
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Won S. Hong
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Rahul R. Telange
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Chee Paul Lin
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. James Bibb
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. David J. Beebe
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Herbert Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Renata Jaskula-Sztul
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jason Whitt.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Whitt, J., Hong, W.S., Telange, R.R. et al. Non-toxic fragment of botulinum neurotoxin type A and monomethyl auristatin E conjugate for targeted therapy for neuroendocrine tumors. Cancer Gene Ther 27, 898–909 (2020). https://doi.org/10.1038/s41417-020-0167-x

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41417-020-0167-x

This article is cited by

Search

Advanced search

Quick links