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Restoration of tumour-growth suppression in vivo via systemic nanoparticle-mediated delivery of PTEN mRNA

An Author Correction to this article was published on 23 November 2018

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Abstract

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a well-characterized tumour-suppressor gene that is lost or mutated in about half of metastatic castration-resistant prostate cancers and in many other human cancers. The restoration of functional PTEN as a treatment for prostate cancer has, however, proven difficult. Here, we show that PTEN messenger RNA (mRNA) can be reintroduced into PTEN-null prostate cancer cells in vitro and in vivo via its encapsulation in polymer–lipid hybrid nanoparticles coated with a polyethylene glycol shell. The nanoparticles are stable in serum, elicit low toxicity and enable high PTEN mRNA transfection in prostate cancer cells. Moreover, significant inhibition of tumour growth is achieved when delivered systemically in multiple mouse models of prostate cancer. We also show that the restoration of PTEN function in PTEN-null prostate cancer cells inhibits the phosphatidylinositol 3-kinase (PI3K)–AKT pathway and enhances apoptosis. Our findings provide proof-of-principle evidence of the restoration of mRNA-based tumour suppression in vivo.

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Fig. 1: Preparation and characterization of mRNA NPs.
Fig. 2: In vitro toxicity and transfection efficiency of mRNA NPs in PC3 cells.
Fig. 3: In vitro mechanism of PTEN-mRNA-PGCP NP treatment in PC3 cells and its therapeutic effect.
Fig. 4: Effect of lipid–PEG on the pharmacokinetics and biodistribution of mRNA NPs.
Fig. 5: In vivo therapeutic validation of PTEN restoration using PTEN-mRNA NPs in a PCa xenograft model.
Fig. 6: In vivo therapeutic validation of PTEN restoration using PTEN-mRNA NPs in a disseminated metastatic PCa model.
Fig. 7: In vivo therapeutic validation of PTEN restoration using PTEN-mRNA NPs in an intratibial orthotopic PCa model.
Fig. 8: In vivo toxicity studies by histopathological and haematological analyses after treatment of mRNA NPs or PBS.

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Data availability

The authors declare that all data supporting the findings of this study are available within the paper and its Supplementary.

Change history

  • 23 November 2018

    The authors wish to add the following sentence into the ‘Competing interests’ section of this Article: “P.W.K. has investment interest in Context Therapeutics LLC, DRGT, Placon, Seer Biosciences and Tarveda Therapeutics, is a company board member for Context Therapeutics LLC, is a consultant and scientific advisory board member for BIND Biosciences, Inc., BN Immunotherapeutics, DRGT, GE Healthcare, Janssen, Metamark, New England Research Institutes, Inc., OncoCellMDX, Progenity, Sanofi, Seer Biosciences, Tarveda Therapeutics and Thermo Fisher, and serves on data safety monitoring boards for Genentech/Roche and Merck.” This has now been included.

References

  1. Song, M. S., Salmena, L. & Pandolfi, P. P. The functions and regulation of the PTEN tumour suppressor. Nat. Rev. Mol. Cell Biol. 13, 283–296 (2012).

    Article  CAS  Google Scholar 

  2. McCall, P., Witton, C. J., Grimsley, S., Nielsen, K. V. & Edwards, J. Is PTEN loss associated with clinical outcome measures in human prostate cancer? Br. J. Cancer 99, 1296–1301 (2008).

    Article  CAS  Google Scholar 

  3. Yoshimoto, M. et al. Interphase FISH analysis of PTEN in histologic sections shows genomic deletions in 68% of primary prostate cancer and 23% of high-grade prostatic intra-epithelial neoplasias. Cancer Genet. Cytogenet. 169, 128–137 (2006).

    Article  CAS  Google Scholar 

  4. Han, B. et al. Fluorescence in situ hybridization study shows association of PTEN deletion with ERG rearrangement during prostate cancer progression. Mod. Pathol. 22, 1083–1093 (2009).

    Article  CAS  Google Scholar 

  5. Verhagen, P. C. et al. The PTEN gene in locally progressive prostate cancer is preferentially inactivated by bi-allelic gene deletion. J. Pathol. 208, 699–707 (2006).

    Article  CAS  Google Scholar 

  6. Yoshimoto, M. et al. FISH analysis of 107 prostate cancers shows that PTEN genomic deletion is associated with poor clinical outcome. Br. J. Cancer. 97, 678–685 (2007).

    Article  CAS  Google Scholar 

  7. Sircar, K. et al. PTEN genomic deletion is associated with p-Akt and AR signalling in poorer outcome, hormone refractory prostate cancer. J. Pathol. 218, 505–513 (2009).

    Article  CAS  Google Scholar 

  8. Schmitz, M. et al. Complete loss of PTEN expression as a possible early prognostic marker for prostate cancer metastasis. Int. J. Cancer 120, 1284–1292 (2007).

    Article  CAS  Google Scholar 

  9. Lotan, T. L. et al. PTEN protein loss by immunostaining: analytic validation and prognostic indicator for a high risk surgical cohort of prostate cancer patients. Clin. Cancer Res. 17, 6563–6573 (2011).

    Article  CAS  Google Scholar 

  10. Stambolic, V. et al. Negative regulation of PKB/Akt-dependent cell survival by the tumour suppressor PTEN. Cell 95, 29–39 (1998).

    Article  CAS  Google Scholar 

  11. Furnari, F. B., Lin, H., Huang, H. S. & Cavenee, W. K. Growth suppression of glioma cells by PTEN requires a functional phosphatase catalytic domain. Proc. Natl Acad. Sci. USA 94, 12479–12484 (1997).

    Article  CAS  Google Scholar 

  12. Sun, H. et al. PTEN modulates cell cycle progression and cell survival by regulating phosphatidylinositol 3,4,5,-trisphosphate and Akt/protein kinase B signaling pathway. Proc. Natl Acad. Sci. USA 96, 6199–6204 (1999).

    Article  CAS  Google Scholar 

  13. Suzuki, A. et al. High cancer susceptibility and embryonic lethality associated with mutation of the PTEN tumour suppressor gene in mice. Curr. Biol. 8, 1169–1178 (1998).

    Article  CAS  Google Scholar 

  14. Maehama, T. & Dixon, J. E. The tumour suppressor, PTEN/MMAC1, dephosphorylates the lipid second messenger, phosphatidylinositol 3,4,5-trisphosphate. J. Biol. Chem. 273, 13375–13378 (1998).

    Article  CAS  Google Scholar 

  15. Engelman, J. A., Luo, J. & Cantley, L. C. The evolution of phosphatidylinositol 3-kinases as regulators of growth and metabolism. Nat. Rev. Genet. 7, 606–619 (2006).

    Article  CAS  Google Scholar 

  16. Taylor, B. S. et al. Integrative genomic profiling of human prostate cancer. Cancer Cell 18, 11–22 (2010).

    Article  CAS  Google Scholar 

  17. Grasso, C. S. et al. The mutational landscape of lethal castration-resistant prostate cancer. Nature 487, 239–243 (2012).

    Article  CAS  Google Scholar 

  18. Backman, S. A. et al. Deletion of Pten in mouse brain causes seizures, ataxia and defects in soma size resembling Lhermitte-Duclos disease. Nat. Genet. 29, 396–403 (2001).

    Article  CAS  Google Scholar 

  19. Liliental, J. et al. Genetic deletion of the Pten tumour suppressor gene promotes cell motility by activation of Rac1 and Cdc42 GTPases. Curr. Biol. 10, 401–404 (2000).

    Article  CAS  Google Scholar 

  20. Tamura, M. et al. Inhibition of cell migration, spreading, and focal adhesions by tumour suppressor PTEN. Science 280, 1614–1617 (1998).

    Article  CAS  Google Scholar 

  21. Hamada, K. et al. The PTEN/PI3K pathway governs normal vascular development and tumour angiogenesis. Genes Dev. 19, 2054–2065 (2005).

    Article  CAS  Google Scholar 

  22. Jiang, B. H. & Liu, L. Z. PI3K/PTEN signaling in angiogenesis and tumourigenesis. Adv. Cancer Res. 102, 19–65 (2009).

    Article  CAS  Google Scholar 

  23. Yin, H. et al. Non-viral vectors for gene-based therapy. Nat. Rev. Genet. 15, 541–555 (2014).

    Article  CAS  Google Scholar 

  24. Quabius, E. S. & Krupp, G. Synthetic mRNAs for manipulating cellular phenotypes: an overview. Nat. Biotechnol. 32, 229–235 (2015).

    CAS  Google Scholar 

  25. Lee, J., Boczkowski, D. & Nair, S. Programming human dendritic cells with mRNA. Methods Mol. Biol. 969, 111–125 (2013).

    Article  CAS  Google Scholar 

  26. Yamamoto, A., Kormann, M., Rosenecker, J. & Rudolph, C. Current prospects for mRNA gene delivery. Eur. J. Pharm. Biopharm. 71, 484–489 (2009).

    Article  CAS  Google Scholar 

  27. Leonhardt, C. et al. Single-cell mRNA transfection studies: delivery, kinetics and statistics by numbers. Nanomedicine 10, 679–688 (2014).

    Article  CAS  Google Scholar 

  28. Ligon, T. S., Leonhardt, C. & Radler, J. O. Multi-level kinetic model of mRNA delivery via transfection of lipoplexes. PLoS ONE 9, e107148 (2014).

    Article  Google Scholar 

  29. Islam, M. A. et al. Biomaterials for mRNA delivery. Biomater. Sci. 3, 1519–1533 (2015).

    Article  CAS  Google Scholar 

  30. Davis, M. E. et al. Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles. Nature 464, 1067–1070 (2010).

    Article  CAS  Google Scholar 

  31. Zuckerman, J. E. et al. Correlating animal and human phase Ia/Ib clinical data with CALAA-01, a targeted, polymer-based nanoparticle containing siRNA. Proc. Natl Acad. Sci. USA 111, 11449–11454 (2014).

    Article  CAS  Google Scholar 

  32. Tabernero, J. et al. First-in-humans trial of an RNA interference therapeutic targeting VEGF and KSP in cancer patients with liver involvement. Cancer Discov. 3, 406–417 (2013).

    Article  CAS  Google Scholar 

  33. Strumberg, D. et al. Phase I clinical development of Atu027, a siRNA formulation targeting PKN3 in patients with advanced solid tumours. Int. J. Clin. Pharmacol. Ther. 50, 76 (2012).

    Article  CAS  Google Scholar 

  34. Schultheis, B. et al. First-in-human phase I study of the liposomal RNA interference therapeutic Atu027 in patients with advanced solid tumours. J. Clin. Oncol. 32, 4141–4148 (2014).

    Article  CAS  Google Scholar 

  35. Tolcher, A. W. et al. A phase 1 study of the BCL2-targeted deoxyribonucleic acid inhibitor (DNAi) PNT2258 in patients with advanced solid tumours. Cancer Chemother. Pharmacol. 73, 363–371 (2014).

    Article  CAS  Google Scholar 

  36. Akinc, A. et al. A combinatorial library of lipid-like materials for delivery of RNAi therapeutics. Nat. Biotechnol. 26, 561–569 (2008).

    Article  CAS  Google Scholar 

  37. Whitehead, K. A. et al. Degradable lipid nanoparticles with predictable in vivo siRNA delivery activity. Nat. Commun. 5, 4277 (2014).

    Article  CAS  Google Scholar 

  38. Whitehead, K. A., Langer, R. & Anderson, D. G. Knocking down barriers: advances in siRNA delivery. Nat. Rev. Drug Discov. 8, 129–138 (2009).

    Article  CAS  Google Scholar 

  39. Zuckerman, J. E. & Davis, M. E. Clinical experiences with systemically administered siRNA-based therapeutics in cancer. Nat. Rev. Drug Discov. 14, 843–856 (2015).

    Article  CAS  Google Scholar 

  40. Conde, J., Oliva, N., Atilano, M., Song, H. S. & Artzi, N. Self-assembled RNA-triple-helix hydrogel scaffold for microRNA modulation in the tumour microenvironment. Nat. Mater. 15, 353–363 (2016).

    Article  CAS  Google Scholar 

  41. Kauffman, K. J. et al. Optimization of lipid nanoparticle formulations for mRNA delivery in vivo with fractional factorial and definitive screening designs. Nano Lett. 15, 7300–7306 (2015).

    Article  CAS  Google Scholar 

  42. Kormann, M. S. et al. Expression of therapeutic proteins after delivery of chemically modified mRNA in mice. Nat. Biotechnol. 29, 154–157 (2011).

    Article  CAS  Google Scholar 

  43. Li, B. et al. An orthogonal array optimization of lipid-like nanoparticles for mRNA delivery in vivo. Nano Lett. 15, 8099–8107 (2015).

    Article  CAS  Google Scholar 

  44. Zhu, X. et al. Long-circulating siRNA nanoparticles for validating Prohibitin1-targeted non-small cell lung cancer treatment. Proc. Natl Acad. Sci. USA 112, 7779–7784 (2015).

    Article  CAS  Google Scholar 

  45. Islam, M. A. et al. The role of osmotic polysorbitol-based transporter in RNAi silencing via caveolae-mediated endocytosis and COX-2 expression. Biomaterials 33, 8868–8880 (2012).

    Article  CAS  Google Scholar 

  46. Islam, M. A. et al. Accelerated gene transfer through a polysorbitol-based transporter mechanism. Biomaterials 32, 9908–9924 (2011).

    Article  CAS  Google Scholar 

  47. Warren, L. et al. Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell 7, 618–630 (2010).

    Article  CAS  Google Scholar 

  48. Wang, Y. et al. Systemic delivery of modified mRNA encoding herpes simplex virus 1 thymidine kinase for targeted cancer gene therapy. Mol. Ther. 21, 358–367 (2013).

    Article  CAS  Google Scholar 

  49. Luo, X. et al. Dual-functional lipid-like nanoparticles for delivery of mRNA and MRI contrast agents. Nanoscale 9, 1575–1579 (2017).

    Article  CAS  Google Scholar 

  50. Huang, H. et al. PTEN induces chemosensitivity in PTEN-mutated prostate cancer cells by suppression of Bcl-2 expression. J. Biol. Chem. 276, 38830–38836 (2001).

    Article  CAS  Google Scholar 

  51. Sturge, J., Caley, M. P. & Waxman, J. Bone metastasis in prostate cancer: emerging therapeutic strategies. Nat. Rev. Clin. Oncol. 8, 357–368 (2011).

    Article  CAS  Google Scholar 

  52. Smukste, I. & Stockwell, B. R. Restoring functions of tumour suppressors with small molecules. Cancer Cell 4, 419–420 (2003).

    Article  CAS  Google Scholar 

  53. Guo, X. E., Ngo, B., Modrek, A. S. & Lee, W. H. Targeting tumour suppressor networks for cancer therapeutics. Curr. Drug Targets 15, 2–16 (2014).

    Article  CAS  Google Scholar 

  54. Bettinger, T., Carlisle, R. C., Read, M. L., Ogris, M. & Seymour, L. W. Peptide-mediated RNA delivery: a novel approach for enhanced transfection of primary and post-mitotic cells. Nucleic Acids Res. 29, 3882–3891 (2001).

    Article  CAS  Google Scholar 

  55. Rejman, J., Tavernier, G., Bavarsad, N., Demeester, J. & De Smedt, S. C. mRNA transfection of cervical carcinoma and mesenchymal stem cells mediated by cationic carriers. J. Control. Release 147, 385–391 (2010).

    Article  CAS  Google Scholar 

  56. Zou, S., Scarfo, K., Nantz, M. H. & Hecker, J. G. Lipid-mediated delivery of RNA is more efficient than delivery of DNA in non-dividing cells. Int. J. Pharm. 389, 232–243 (2010).

    Article  CAS  Google Scholar 

  57. Read, M. L. et al. A versatile reducible polycation-based system for efficient delivery of a broad range of nucleic acids. Nucleic Acids Res. 33, e86 (2005).

    Article  Google Scholar 

  58. Kong, G., Braun, R. D. & Dewhirst, M. W. Hyperthermia enables tumour-specific nanoparticle delivery: effect of particle size. Cancer Res. 60, 4440–4445 (2000).

    CAS  PubMed  Google Scholar 

  59. Alexis, F., Pridgen, E., Molnar, L. K. & Farokhzad, O. C. Factors affecting the clearance and biodistribution of polymeric nanoparticles. Mol. Pharm. 5, 505–515 (2008).

    Article  CAS  Google Scholar 

  60. Shi, J., Kantoff, P. W., Wooster, R. & Farokhzad, O. C. Cancer nanomedicine: progress, challenges and opportunities. Nat. Rev. Cancer 17, 20–37 (2017).

    Article  CAS  Google Scholar 

  61. Bertrand, N., Wu, J., Xu, X., Kamaly, N. & Farokhzad, O. C. Cancer nanotechnology: the impact of passive and active targeting in the era of modern cancer biology. Adv. Drug Deliv. Rev. 66, 2–25 (2014).

    Article  CAS  Google Scholar 

  62. Knop, K., Hoogenboom, R., Fischer, D. & Schubert, U. S. Poly(ethylene glycol) in drug delivery: pros and cons as well as potential alternatives. Angew. Chem. Int. Ed. Engl. 49, 6288–6308 (2010).

    Article  CAS  Google Scholar 

  63. Guo, X. & Huang, L. Recent advances in nonviral vectors for gene delivery. Acc. Chem. Res. 45, 971–979 (2012).

    Article  CAS  Google Scholar 

  64. Liu, H. et al. Structure-based programming of lymph-node targeting in molecular vaccines. Nature 507, 519–522 (2014).

    Article  CAS  Google Scholar 

  65. Li, J. et al. PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer. Science 275, 1943–1947 (1997).

    Article  CAS  Google Scholar 

  66. Steck, P. A. et al. Identification of a candidate tumour suppressor gene, MMAC1, at chromosome 10q23.3 that is mutated in multiple advanced cancers. Nat. Genet. 15, 356–362 (1997).

    Article  CAS  Google Scholar 

  67. Di Cristofano, A., De Acetis, M., Koff, A., Cordon-Cardo, C. & Pandolfi, P. P. Pten and p27KIP1 cooperate in prostate cancer tumour suppression in the mouse. Nat. Genet. 27, 222–224 (2001).

    Article  Google Scholar 

  68. Hopkins, B. D. et al. A secreted PTEN phosphatase that enters cells to alter signaling and survival. Science 341, 399–402 (2013).

    Article  CAS  Google Scholar 

  69. Masson, G. R., Perisic, O., Burke, J. E. & Williams, R. L. The intrinsically disordered tails of PTEN and PTEN-L have distinct roles in regulating substrate specificity and membrane activity. Biochem. J. 473, 135–144 (2016).

    Article  CAS  Google Scholar 

  70. Juric, D. et al. Convergent loss of PTEN leads to clinical resistance to a PI(3)Kalpha inhibitor. Nature 518, 240–244 (2015).

    Article  CAS  Google Scholar 

  71. Peng, W. et al. Loss of PTEN promotes resistance to T cell-mediated immunotherapy. Cancer Discov 6, 202–216 (2016).

    Article  CAS  Google Scholar 

  72. Campeau, E. et al. A versatile viral system for expression and depletion of proteins in mammalian cells. PLoS ONE 4, e6529 (2009).

    Article  Google Scholar 

  73. Ramaswamy, S. et al. Regulation of G1 progression by the PTEN tumour suppressor protein is linked to inhibition of the phosphatidylinositol 3-kinase/Akt pathway. Proc. Natl Acad. Sci. USA 96, 2110–2115 (1999).

    Article  CAS  Google Scholar 

  74. Xu, X. et al. Enhancing tumour cell response to chemotherapy through nanoparticle-mediated codelivery of siRNA and cisplatin prodrug. Proc. Natl Acad. Sci. USA 110, 18638–18643 (2013).

    Article  CAS  Google Scholar 

  75. Cox, T. R. et al. The hypoxic cancer secretome induces pre-metastatic bone lesions through lysyl oxidase. Nature 522, 106–110 (2015).

    Article  CAS  Google Scholar 

  76. Krzywinski, M. & Altman, N. Points of significance: nonparametric tests. Nat. Methods 11, 467–468 (2014).

    Article  CAS  Google Scholar 

  77. Tammela, T. et al. A Wnt-producing niche drives proliferative potential and progression in lung adenocarcinoma. Nature 545, 355–359 (2017).

    Article  CAS  Google Scholar 

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Acknowledgements

This work was in part supported by the Prostate Cancer Foundation (PCF) Young Investigator Award (J.S.); the David Koch-PCF Award in Nanotherapeutics (O.C.F., R.L. and P.W.K.); the US National Institutes of Health (NIH) grants CA200900 (J.S. and B.R.Z.), HL127464 (O.C.F.) and R00CA160350 (J.S.); the National Research Foundation of Korea (K1A1A2048701) (O.C.F.); and the DoD Prostate Cancer Research Program Postdoctoral Training Award (W81XWH-14-1-0268) (Y.X.). The authors would also like to thank E. Reesor, S. Guillemette, J. Rice, Y. Li, M. Zaffagni, D. Bielenberg and J. Wang for their assistance. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

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M.A.I., Y.X., O.C.F., B.R.Z. and J.S. conceived the idea, designed the study and directed the project. M.A.I., Y.X. and W.T. performed all the experiments and analysed data. W.T., J.M.U., K.Z. and G.Y.L. assisted with the metastatic and orthotopic PCa experiments in vivo. M.L., D.A., J.T.O. and W.C. helped in nanoparticle preparation and experimental assays. R.L. provided reagents and conceptual advice. W.T., H.Z., M.Y., M.D., M.M., P.W.K. provided technical support and corrections of manuscript. M.A.I., Y.X. and W.T. wrote the manuscript and revised according to the comments of R.L., P.W.K., O.C.F., B.R.Z. and J.S.

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Correspondence to Omid C. Farokhzad, Bruce R. Zetter or Jinjun Shi.

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O.C.F. and R.L. declare financial interests in Selecta Biosciences, Tarveda Therapeutics, Placon Therapeutics and Seer. R.L. declares financial interests in Moderna Therapeutics. P.W.K. has investment interest in Context Therapeutics LLC, DRGT, Placon, Seer Biosciences and Tarveda Therapeutics, is a company board member for Context Therapeutics LLC, is a consultant and scientific advisory board member for BIND Biosciences, Inc., BN Immunotherapeutics, DRGT, GE Healthcare, Janssen, Metamark, New England Research Institutes, Inc., OncoCellMDX, Progenity, Sanofi, Seer Biosciences, Tarveda Therapeutics and Thermo Fisher, and serves on data safety monitoring boards for Genentech/Roche and Merck.

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Islam, M.A., Xu, Y., Tao, W. et al. Restoration of tumour-growth suppression in vivo via systemic nanoparticle-mediated delivery of PTEN mRNA. Nat Biomed Eng 2, 850–864 (2018). https://doi.org/10.1038/s41551-018-0284-0

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