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AAV-mediated expression of 3TSR inhibits tumor and metastatic lesion development and extends survival in a murine model of epithelial ovarian carcinoma

Abstract

An integral step in the development of solid tumors is the recruitment of blood vessels to fuel tumor growth. Antiangiogenic therapies can inhibit this process and control solid tumor growth. Thrombospondin-1 is an antiangiogenic protein possessing three type I repeats (3TSR) near the center of the protein and a CD47-binding peptide (CD47) in its C-terminus. Previously, we showed that treatment with recombinant 3TSR induces tumor regression, normalizes tumor vasculature, and improves uptake of chemotherapy drugs in an orthotopic, syngeneic mouse model of advanced stage epithelial ovarian cancer (EOC). While effective, this intervention required daily intraperitoneal injections. To circumvent this, here we employ adeno-associated virus (AAV) gene therapy vectors to express 3TSR alone or in combination with the CD47-binding peptide of TSP-1 and evaluate the impact on tumor development and survival in a mouse model of EOC. A single intraperitoneal injection of 1 × 1011 vg of AAV expressing 3TSR, CD47-binding peptide, or 3TSR + CD47 effectively suppressed primary tumor growth; however, only AAV-3TSR was able to inhibit development of secondary lesions at 90-days post-tumor implantation and significantly improve survival. Taken together, AAV-mediated expression of 3TSR appears safe and effective at inhibiting tumor development and represents a novel, less invasive approach for treating ovarian carcinoma.

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References

  1. 1.

    NCI. Surveillance, epidemiology, and end results (SEER). National Cancer Institute; 2018.

  2. 2.

    Agarwal R, Kaye SB. Ovarian cancer: strategies for overcoming resistance to chemotherapy. Nat Rev Cancer. 2003;3:502–16.

  3. 3.

    Sato-Dahlman M, Wirth K, Yamamoto M. Role of gene therapy in pancreatic cancer-a review. Cancers. 2018;10:E103.

  4. 4.

    Schambach A, Morgan M. Retroviral vectors for cancer gene therapy. Recent Results Cancer Res. 2016;209:17–35.

  5. 5.

    Lin T, Zhang L, Davis J, Gu J, Nishizaki M, Ji L, et al. Combination of TRAIL gene therapy and chemotherapy enhances antitumor and antimetastasis effects in chemosensitive and chemoresistant breast cancers. Mol Ther. 2003;8:441–8.

  6. 6.

    Li ZB, Zeng ZJ, Chen Q, Luo SQ, Hu WX. Recombinant AAV-mediated HSVtk gene transfer with direct intratumoral injections and Tet-On regulation for implanted human breast cancer. BMC Cancer. 2006;6:66.

  7. 7.

    Lengyel E. Ovarian cancer development and metastasis. Am J Pathol. 2010;177:1053–64.

  8. 8.

    Xie Y, Hicks MJ, Kaminsky SM, Moore MA, Crystal RG, Rafii A. AAV-mediated persistent bevacizumab therapy suppresses tumor growth of ovarian cancer. Gynecol Oncol. 2014;135:325–32.

  9. 9.

    Good DJ, Polverini PJ, Rastinejad F, Le Beau MM, Lemons RS, Frazier WA, et al. A tumor suppressor-dependent inhibitor of angiogenesis is immunologically and functionally indistinguishable from a fragment of thrombospondin. Proc Natl Acad Sci USA. 1990;87:6624–8.

  10. 10.

    Henkin J, Volpert OV. Therapies using anti-angiogenic peptide mimetics of thrombospondin-1. Expert Opin Ther Targets. 2011;15:1369–86.

  11. 11.

    Miao WM, Seng WL, Duquette M, Lawler P, Laus C, Lawler J. Thrombospondin-1 type 1 repeat recombinant proteins inhibit tumor growth through transforming growth factor-beta-dependent and -independent mechanisms. Cancer Res. 2001;61:7830–9.

  12. 12.

    Greenaway J, Henkin J, Lawler J, Moorehead R, Petrik J. ABT-510 induces tumor cell apoptosis and inhibits ovarian tumor growth in an orthotopic, syngeneic model of epithelial ovarian cancer. Mol Cancer Ther. 2009;8:64–74.

  13. 13.

    Campbell N, Greenaway J, Henkin J, Petrik J. ABT-898 induces tumor regression and prolongs survival in a mouse model of epithelial ovarian cancer. Mol Cancer Ther. 2011;10:1876–85.

  14. 14.

    Campbell NE, Greenaway J, Henkin J, Moorehead RA, Petrik J. The thrombospondin-1 mimetic ABT-510 increases the uptake and effectiveness of cisplatin and paclitaxel in a mouse model of epithelial ovarian cancer. Neoplasia. 2010;12:275–83.

  15. 15.

    Russell S, Duquette M, Liu J, Drapkin R, Lawler J, Petrik J. Combined therapy with thrombospondin-1 type I repeats (3TSR) and chemotherapy induces regression and significantly improves survival in a preclinical model of advanced stage epithelial ovarian cancer. FASEB J. 2015;29:576–88.

  16. 16.

    Matuszewska K, Santry LA, van Vloten JP, Au Yeung AW, Major PP, Lawler J, et al. Combining vascular normalization with an oncolytic virus enhances immunotherapy in a preclinical model of advanced-stage ovarian cancer. Clin Cancer Res. 2018;25:1624–38.

  17. 17.

    Chao MP, Weissman IL, Majeti R. The CD47-SIRPα pathway in cancer immune evasion and potential therapeutic implications. Curr Opin Immunol. 2012;24:225–32.

  18. 18.

    Kaur S, Martin-Manso G, Pendrak ML, Garfield SH, Isenberg JS, Roberts DD. Thrombospondin-1 inhibits VEGF receptor-2 signaling by disrupting its association with CD47. J Biol Chem. 2010;285:38923–32.

  19. 19.

    Bertolini F, Paul S, Mancuso P, Monestiroli S, Gobbi A, Shaked Y, et al. Maximum tolerable dose and low-dose metronomic chemotherapy have opposite effects on the mobilization and viability of circulating endothelial progenitor cells. Cancer Res. 2003;63:4342–6.

  20. 20.

    Pietras K, Hanahan D. A multitargeted, metronomic, and maximum-tolerated dose “chemo-switch” regimen is antiangiogenic, producing objective responses and survival benefit in a mouse model of cancer. J Clin Oncol. 2005;23:939–52.

  21. 21.

    Roby KF, Taylor CC, Sweetwood JP, Cheng Y, Pace JL, Tawfik O, et al. Development of a syngeneic mouse model for events related to ovarian cancer. Carcinogenesis. 2000;21:585–91.

  22. 22.

    Kim JH, Lee SR, Li LH, Park HJ, Park JH, Lee KY, et al. High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. PLoS ONE. 2011;6:e18556.

  23. 23.

    Gerhard DS, Wagner L, Feingold EA, Shenmen CM, Grouse LH, Schuler G, et al. The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC). Genome Res. 2004;14:2121–7.

  24. 24.

    Halbert CL, Allen JM, Chamberlain JS. AAV6 vector production and purification for muscle gene therapy. Methods Mol Biol. 2018;1687:257–66.

  25. 25.

    van Lieshout LP, Domm JM, Wootton SK. AAV-mediated gene delivery to the lung. Methods Mol Biol. 2019;1950:361–72.

  26. 26.

    Huang X, Hartley AV, Yin Y, Herskowitz JH, Lah JJ, Ressler KJ. AAV2 production with optimized N/P ratio and PEI-mediated transfection results in low toxicity and high titer for in vitro and in vivo applications. J Virol Methods. 2013;193:270–7.

  27. 27.

    Aurnhammer C, Haase M, Muether N, Hausl M, Rauschhuber C, Huber I, et al. Universal real-time PCR for the detection and quantification of adeno-associated virus serotype 2-derived inverted terminal repeat sequences. Hum Gene Ther Methods. 2012;23:18–28.

  28. 28.

    Linnerth-Petrik NM, Santry LA, Petrik JJ, Wootton SK. Opposing functions of Akt isoforms in lung tumor initiation and progression. PLoS ONE. 2014;9:e94595.

  29. 29.

    Greenaway J, Moorehead R, Shaw P, Petrik J. Epithelial-stromal interaction increases cell proliferation, survival and tumorigenicity in a mouse model of human epithelial ovarian cancer. Gynecol Oncol. 2008;108:385–94.

  30. 30.

    Yan J, Wang H, Xu Q, Jain N, Toxavidis V, Tigges J, et al. Signal sequence is still required in genes downstream of “autocleaving” 2A peptide for secretary or membrane-anchored expression. Anal Biochem. 2010;399:144–6.

  31. 31.

    Lawler J, Detmar M. Tumor progression: the effects of thrombospondin-1 and -2. Int J Biochem Cell Biol. 2004;36:1038–45.

  32. 32.

    Zhang X, Xu J, Lawler J, Terwilliger E, Parangi S. Adeno-associated virus-mediated antiangiogenic gene therapy with thrombospondin-1 type 1 repeats and endostatin. Clin Cancer Res. 2007;13:3968–76.

  33. 33.

    Short SM, Derrien A, Narsimhan RP, Lawler J, Ingber DE, Zetter BR. Inhibition of endothelial cell migration by thrombospondin-1 type-1 repeats is mediated by beta1 integrins. J Cell Biol. 2005;168:643–53.

  34. 34.

    Zhang X, Galardi E, Duquette M, Lawler J, Parangi S. Antiangiogenic treatment with three thrombospondin-1 type 1 repeats versus gemcitabine in an orthotopic human pancreatic cancer model. Clin Cancer Res. 2005;11:5622–30.

  35. 35.

    Kisker O, Becker CM, Prox D, Fannon M, D’Amato R, Flynn E, et al. Continuous administration of endostatin by intraperitoneally implanted osmotic pump improves the efficacy and potency of therapy in a mouse xenograft tumor model. Cancer Res. 2001;61:7669–74.

  36. 36.

    Drixler TA, Borel Rinkes IH, Ritchie ED, van Vroonhoven TJ, Gebbink MF, Voest EE. Continuous administration of angiostatin inhibits accelerated growth of colorectal liver metastases after partial hepatectomy. Cancer Res. 2000;60:1761–5.

  37. 37.

    Zhang X, Connolly C, Duquette M, Lawler J, Parangi S. Continuous administration of the three thrombospondin-1 type 1 repeats recombinant protein improves the potency of therapy in an orthotopic human pancreatic cancer model. Cancer Lett. 2007;247:143–9.

  38. 38.

    Hastie E, Samulski RJ. Adeno-associated virus at 50: a golden anniversary of discovery, research, and gene therapy success–a personal perspective. Hum Gene Ther. 2015;26:257–65.

  39. 39.

    Matuszewska K, Santry LA, van Vloten JP, Au Yeung AWK, Major PP, Lawler J et al. Combining vascular normalization with an oncolytic virus enhances immunotherapy in a preclinical model of advanced-stage ovarian cancer. Clinical Cancer Res. 2018.

  40. 40.

    Chtarto A, Bender HU, Hanemann CO, Kemp T, Lehtonen E, Levivier M, et al. Tetracycline-inducible transgene expression mediated by a single AAV vector. Gene Ther. 2003;10:84–94.

  41. 41.

    Lee TY, Tjin Tham Sjin RM, Movahedi S, Ahmed B, Pravda EA, Lo KM, et al. Linking antibody Fc domain to endostatin significantly improves endostatin half-life and efficacy. Clin Cancer Res. 2008;14:1487–93.

  42. 42.

    Wang L, Nichols TC, Read MS, Bellinger DA, Verma IM. Sustained expression of therapeutic level of factor IX in hemophilia B dogs by AAV-mediated gene therapy in liver. Mol Ther. 2000;1:154–8.

  43. 43.

    Crudele JM, Finn JD, Siner JI, Martin NB, Niemeyer GP, Zhou S, et al. AAV liver expression of FIX-Padua prevents and eradicates FIX inhibitor without increasing thrombogenicity in hemophilia B dogs and mice. Blood. 2015;125:1553–61.

  44. 44.

    Sabatino DE, Lange AM, Altynova ES, Sarkar R, Zhou S, Merricks EP, et al. Efficacy and safety of long-term prophylaxis in severe hemophilia A dogs following liver gene therapy using AAV vectors. Mol Ther. 2011;19:442–9.

  45. 45.

    van Lieshout LP, Soule G, Sorensen D, Frost KL, He S, Tierney K, et al. Intramuscular adeno-associated virus-mediated expression of monoclonal antibodies provides 100% protection against ebola virus infection in mice. J Infect Dis. 2018;217:916–25.

  46. 46.

    Song S, Morgan M, Ellis T, Poirier A, Chesnut K, Wang J, et al. Sustained secretion of human alpha-1-antitrypsin from murine muscle transduced with adeno-associated virus vectors. Proc Natl Acad Sci USA. 1998;95:14384–8.

  47. 47.

    Kessler PD, Podsakoff GM, Chen X, McQuiston SA, Colosi PC, Matelis LA, et al. Gene delivery to skeletal muscle results in sustained expression and systemic delivery of a therapeutic protein. Proc Natl Acad Sci USA. 1996;93:14082–7.

  48. 48.

    Buchlis G, Podsakoff GM, Radu A, Hawk SM, Flake AW, Mingozzi F, et al. Factor IX expression in skeletal muscle of a severe hemophilia B patient 10 years after AAV-mediated gene transfer. Blood. 2012;119:3038–41.

  49. 49.

    Zincarelli C, Soltys S, Rengo G, Rabinowitz J. Analysis of AAV serotypes 1-9 mediated gene expression and tropism in mice after systemic injection. Mol Ther. 2008;16:1073–80.

  50. 50.

    Wu Z, Asokan A, Grieger JC, Govindasamy L, Agbandje-McKenna M, Samulski RJ. Single amino acid changes can influence titer, heparin binding, and tissue tropism in different adeno-associated virus serotypes. J Virol. 2006;80:11393–7.

  51. 51.

    Mao Y, Wang X, Yan R, Hu W, Li A, Wang S, et al. Single point mutation in adeno-associated viral vectors -DJ capsid leads to improvement for gene delivery in vivo. BMC Biotechnol. 2016;16:1.

  52. 52.

    van Lieshout LP, Domm JM, Rindler TN, Frost KL, Sorensen DL, Medina SJ, et al. A novel triple-mutant AAV6 capsid induces rapid and potent transgene expression in the muscle and respiratory tract of mice. Mol Ther Methods Clin Dev. 2018;9:323–9.

  53. 53.

    Rivera VM, Gao GP, Grant RL, Schnell MA, Zoltick PW, Rozamus LW, et al. Long-term pharmacologically regulated expression of erythropoietin in primates following AAV-mediated gene transfer. Blood. 2005;105:1424–30.

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Acknowledgements

We thank Campus Animal Facilities, University of Guelph, for animal care services. We also thank Betty-Anne McBey for her technical assistance with this project and Jessica Minott for assistance with monitoring mice during the survival study.

Funding

Funding for this research was provided by grants from the Canadian Institutes of Health Research (JJP) and the Cancer Research Society (JJP and SKW) AAS was supported by a Vanier Canada Graduate Scholarship (CIHR) and a Brock Doctoral Scholarship; DLY was supported by an OVC PhD Scholarship. The project was also supported by a CAO Pilot grant from the Beth Israel Deaconess Medical Center (JL).

Author information

Correspondence to Byram W. Bridle or James J. Petrik or Sarah K. Wootton.

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Conflict of interest

JJP and JL are co-inventors on US patent US20140271641A1 for the treatment of ovarian cancer with 3TSR.

Ethics

All animal experiments were conducted in accordance with the Canadian Council on Animal Care guidelines and approved by the Animal Care Committee of the University of Guelph (AUP# 3827). 8-week-old C57BL/6 female mice were purchased from Charles River Laboratories (St. Constant, Quebec, Canada).

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