Journal of Molecular Medicine

, Volume 97, Issue 5, pp 691–709 | Cite as

PRSS21/testisin inhibits ovarian tumor metastasis and antagonizes proangiogenic angiopoietins ANG2 and ANGPTL4

  • Gregory D. Conway
  • Marguerite S. Buzza
  • Erik W. Martin
  • Nadire Duru
  • Tierra A. Johnson
  • Raymond J. Peroutka
  • Nisha R. Pawar
  • Toni M. AntalisEmail author
Original Article


Ovarian cancer is the leading cause of death among all the gynecological cancers in the USA. Ovarian cancer employs a unique mode of metastasis, as exfoliated tumor cells disseminate within the peritoneal cavity, colonizing in several sites as well as accumulating ascites. Tumor recurrence and widespread metastasis are significant factors contributing to poor prognosis. PRSS21 is a metastasis-associated ovarian cancer gene that encodes the glycosyl-phosphatidylinositol-linked serine protease, testisin. Testisin expression is increased in multiple ovarian tumor types, with relatively little expression in normal tissues, but is differentially decreased in metastatic ovarian serous carcinomas compared to primary tumors. Here we explored the function of testisin in late-stage ovarian cancer progression using a murine xenograft model of ovarian intraperitoneal tumor metastasis. Increased tumor testisin expression inhibited intra-peritoneal tumor seeding and colonization, ascites accumulation, and metastatic tumor burden that was dependent on catalytically active testisin. The known testisin substrate, protease-activated receptor-2 (PAR-2), is a target of testisin activity. Gene profiling and mechanistic studies demonstrate that testisin activity suppresses the synthesis and secretion of pro-angiogenic angiopoietins, ANG2 and ANGPTL4, which normally promote vascular leak and edema. These observations support a model wherein testisin activates PAR-2 to antagonize proangiogenic angiopoietins that modulate vascular permeability and ascites accumulation associated with ovarian tumor metastasis.

Key messages

  • Testisin inhibits metastatic ovarian tumor burden and ascites production.

  • Testisin activity antagonizes ANG2 and ANGPTL4 synthesis and secretion.

  • PAR-2 is a proteolytic target of testisin on the surface of ovarian cancer cells.


Testisin Ovarian metastasis ANG2 Ascites Serine protease 



We thank Eun Yong Choi, M.D., and Rena Lapidus, Ph.D., of the Translational Laboratory Shared Services, University of Maryland School of Medicine, for assistance with the in vivo tumor studies and the in vivo bioluminescence imaging.

Funding information

This work was supported by the National Institutes of Health Grants R01 CA196988, R01 HL118390 and T32 CA154274.

Compliance with ethical standards

All procedures performed in studies involving animals were in accordance with the ethical standards of the University of Maryland School of Medicine Institutional Animal Care and Use Committee (IACUC), the institution at which the studies were conducted.

Conflict of interest

The authors declare that they have no competing interests.

Supplementary material

109_2019_1763_MOESM1_ESM.pdf (2.7 mb)
ESM 1 (PDF 2.71 mb)


  1. 1.
    Bast RC Jr, Hennessy B, Mills GB (2009) The biology of ovarian cancer: new opportunities for translation. Nat Rev Cancer 9:415–428CrossRefGoogle Scholar
  2. 2.
    Lengyel E (2010) Ovarian cancer development and metastasis. Am J Pathol 177:1053–1064CrossRefGoogle Scholar
  3. 3.
    Bamias A, Pignata S, Pujade-Lauraine E (2012) Angiogenesis: a promising therapeutic target for ovarian cancer. Crit Rev Oncol Hematol 84:314–326CrossRefGoogle Scholar
  4. 4.
    Ramakrishnan S, Subramanian IV, Yokoyama Y, Geller M (2005) Angiogenesis in normal and neoplastic ovaries. Angiogenesis 8:169–182CrossRefGoogle Scholar
  5. 5.
    Eskander RN, Tewari KS (2012) Emerging treatment options for management of malignant ascites in patients with ovarian cancer. Int J Women's Health 4:395–404Google Scholar
  6. 6.
    Herr D, Sallmann A, Bekes I, Konrad R, Holzheu I, Kreienberg R, Wulff C (2012) VEGF induces ascites in ovarian cancer patients via increasing peritoneal permeability by downregulation of Claudin 5. Gynecol Oncol 127:210–216CrossRefGoogle Scholar
  7. 7.
    Shigemasa K, Underwood LJ, Beard J, Tanimoto H, Ohama K, Parmley TH, O'Brien TJ (2000) Overexpression of testisin, a serine protease expressed by testicular germ cells, in epithelial ovarian tumor cells. J Soc Gynecol Investig 7:358–362Google Scholar
  8. 8.
    Aimes RT, Zijlstra A, Hooper JD, Ogbourne SM, Sit ML, Fuchs S, Gotley DC, Quigley JP, Antalis TM (2003) Endothelial cell serine proteases expressed during vascular morphogenesis and angiogenesis. Thromb Haemost 89:561–572CrossRefGoogle Scholar
  9. 9.
    Hooper JD, Bowen N, Marshall H, Cullen LM, Sood R, Daniels R, Stuttgen MA, Normyle JF, Higgs DR, Kastner DL, Ogbourne SM, Pera MF, Jazwinska EC, Antalis TM (2000) Localization, expression and genomic structure of the gene encoding the human serine protease testisin. Biochim Biophys Acta 1492:63–71CrossRefGoogle Scholar
  10. 10.
    Hooper JD, Nicol DL, Dickinson JL, Eyre HJ, Scarman AL, Normyle JF, Stuttgen MA, Douglas ML, Loveland KA, Sutherland GR, Antalis TM (1999) Testisin, a new human serine proteinase expressed by premeiotic testicular germ cells and lost in testicular germ cell tumors. Cancer Res 59:3199–3205Google Scholar
  11. 11.
    Scarman AL, Hooper JD, Boucaut KJ, Sit ML, Webb GC, Normyle JF, Antalis TM (2001) Organization and chromosomal localization of the murine testisin gene encoding a serine protease temporally expressed during spermatogenesis. Eur J Biochem 268:1250–1258CrossRefGoogle Scholar
  12. 12.
    Netzel-Arnett S, Bugge TH, Hess RA, Carnes K, Stringer BW, Scarman AL, Hooper JD, Tonks ID, Kay GF, Antalis TM (2009) The glycosylphosphatidylinositol-anchored serine protease PRSS21 (testisin) imparts murine epididymal sperm cell maturation and fertilizing ability. Biol Reprod 81:921–932CrossRefGoogle Scholar
  13. 13.
    Tang T, Kmet M, Corral L, Vartanian S, Tobler A, Papkoff J (2005) Testisin, a glycosyl-phosphatidylinositol-linked serine protease, promotes malignant transformation in vitro and in vivo. Cancer Res 65:868–878Google Scholar
  14. 14.
    Bignotti E, Tassi RA, Calza S, Ravaggi A, Bandiera E, Rossi E, Donzelli C, Pasinetti B, Pecorelli S, Santin AD (2007) Gene expression profile of ovarian serous papillary carcinomas: identification of metastasis-associated genes. Am J Obstet Gynecol 196:245 e241–245 e211CrossRefGoogle Scholar
  15. 15.
    Driesbaugh KH, Buzza MS, Martin EW, Conway GD, Kao JP, Antalis TM (2015) Proteolytic activation of the protease-activated receptor (PAR)-2 by the glycosylphosphatidylinositol-anchored serine protease testisin. J Biol Chem 290:3529–3541CrossRefGoogle Scholar
  16. 16.
    Adams MN, Ramachandran R, Yau MK, Suen JY, Fairlie DP, Hollenberg MD, Hooper JD (2011) Structure, function and pathophysiology of protease activated receptors. Pharmacol Ther 130:248–282CrossRefGoogle Scholar
  17. 17.
    Jahan I, Fujimoto J, Alam SM, Sato E, Sakaguchi H, Tamaya T (2007) Role of protease activated receptor-2 in tumor advancement of ovarian cancers. Ann Oncol 18:1506–1512CrossRefGoogle Scholar
  18. 18.
    Ruf W, Yokota N, Schaffner F (2010) Tissue factor in cancer progression and angiogenesis. Thromb Res 125(Suppl 2):S36–S38CrossRefGoogle Scholar
  19. 19.
    Chanakira A, Westmark PR, Ong IM, Sheehan JP (2017) Tissue factor-factor VIIa complex triggers protease activated receptor 2-dependent growth factor release and migration in ovarian cancer. Gynecol Oncol 145:167–175CrossRefGoogle Scholar
  20. 20.
    Luo R, Wang X, Dong Y, Wang L, Tian C (2014) Activation of protease-activated receptor 2 reduces glioblastoma cell apoptosis. J Biomed Sci 21:25CrossRefGoogle Scholar
  21. 21.
    Iablokov V, Hirota CL, Peplowski MA, Ramachandran R, Mihara K, Hollenberg MD, MacNaughton WK (2014) Proteinase-activated receptor 2 (PAR2) decreases apoptosis in colonic epithelial cells. J Biol Chem 289:34366–34377CrossRefGoogle Scholar
  22. 22.
    Huang SH, Li Y, Chen HG, Rong J, Ye S (2013) Activation of proteinase-activated receptor 2 prevents apoptosis of lung cancer cells. Cancer Investig 31:578–581CrossRefGoogle Scholar
  23. 23.
    Aman M, Ohishi Y, Imamura H, Shinozaki T, Yasutake N, Kato K, Oda Y (2017) Expression of protease-activated receptor-2 (PAR-2) is related to advanced clinical stage and adverse prognosis in ovarian clear cell carcinoma. Hum Pathol 64:156–163CrossRefGoogle Scholar
  24. 24.
    Schaffner F, Yokota N, Ruf W (2012) Tissue factor proangiogenic signaling in cancer progression. Thromb Res 129(Suppl 1):S127–S131CrossRefGoogle Scholar
  25. 25.
    Zhu T, Sennlaub F, Beauchamp MH, Fan L, Joyal JS, Checchin D, Nim S, Lachapelle P, Sirinyan M, Hou X, Bossolasco M, Rivard GE, Heveker N, Chemtob S (2006) Proangiogenic effects of protease-activated receptor 2 are tumor necrosis factor-alpha and consecutively Tie2 dependent. Arterioscler Thromb Vasc Biol 26:744–750CrossRefGoogle Scholar
  26. 26.
    Shaw TJ, Senterman MK, Dawson K, Crane CA, Vanderhyden BC (2004) Characterization of intraperitoneal, orthotopic, and metastatic xenograft models of human ovarian cancer. Mol Ther 10:1032–1042CrossRefGoogle Scholar
  27. 27.
    Rothmeier AS, Ruf W (2012) Protease-activated receptor 2 signaling in inflammation. Semin Immunopathol 34:133–149CrossRefGoogle Scholar
  28. 28.
    Johnson JJ, Miller DL, Jiang R, Liu Y, Shi Z, Tarwater L, Williams R, Balsara R, Sauter ER, Stack MS (2016) Protease-activated Receptor-2 (PAR-2)-mediated Nf-kappaB activation suppresses inflammation-associated tumor suppressor microRNAs in oral squamous cell carcinoma. J Biol Chem 291:6936–6945CrossRefGoogle Scholar
  29. 29.
    Wang Y, Xu RC, Zhang XL, Niu XL, Qu Y, Li LZ, Meng XY (2012) Interleukin-8 secretion by ovarian cancer cells increases anchorage-independent growth, proliferation, angiogenic potential, adhesion and invasion. Cytokine 59:145–155CrossRefGoogle Scholar
  30. 30.
    Lane D, Matte I, Rancourt C, Piche A (2011) Prognostic significance of IL-6 and IL-8 ascites levels in ovarian cancer patients. BMC Cancer 11:210CrossRefGoogle Scholar
  31. 31.
    Tanaka Y, Sekiguchi F, Hong H, Kawabata A (2008) PAR2 triggers IL-8 release via MEK/ERK and PI3-kinase/Akt pathways in GI epithelial cells. Biochem Biophys Res Commun 377:622–626CrossRefGoogle Scholar
  32. 32.
    Shi J, Wei PK (2016) Interleukin-8: a potent promoter of angiogenesis in gastric cancer. Oncol Lett 11:1043–1050CrossRefGoogle Scholar
  33. 33.
    Ning Y, Manegold PC, Hong YK, Zhang W, Pohl A, Lurje G, Winder T, Yang D, LaBonte MJ, Wilson PM et al (2011) Interleukin-8 is associated with proliferation, migration, angiogenesis and chemosensitivity in vitro and in vivo in colon cancer cell line models. Int J Cancer 128:2038–2049CrossRefGoogle Scholar
  34. 34.
    Barry GD, Suen JY, Le GT, Cotterell A, Reid RC, Fairlie DP (2010) Novel agonists and antagonists for human protease activated receptor 2. J Med Chem 53:7428–7440CrossRefGoogle Scholar
  35. 35.
    Manton KJ, Douglas ML, Netzel-Arnett S, Fitzpatrick DR, Nicol DL, Boyd AW, Clements JA, Antalis TM (2005) Hypermethylation of the 5' CpG island of the gene encoding the serine protease testisin promotes its loss in testicular tumorigenesis. Br J Cancer 92:760–769CrossRefGoogle Scholar
  36. 36.
    Hata K, Udagawa J, Fujiwaki R, Nakayama K, Otani H, Miyazaki K (2002) Expression of angiopoietin-1, angiopoietin-2, and Tie2 genes in normal ovary with corpus luteum and in ovarian cancer. Oncology 62:340–348CrossRefGoogle Scholar
  37. 37.
    Zhu P, Tan MJ, Huang RL, Tan CK, Chong HC, Pal M, Lam CR, Boukamp P, Pan JY, Tan SH et al (2011) Angiopoietin-like 4 protein elevates the prosurvival intracellular O2(-):H2O2 ratio and confers anoikis resistance to tumors. Cancer Cell 19:401–415CrossRefGoogle Scholar
  38. 38.
    Sallinen H, Heikura T, Laidinen S, Kosma VM, Heinonen S, Yla-Herttuala S, Anttila M (2010) Preoperative angiopoietin-2 serum levels: a marker of malignant potential in ovarian neoplasms and poor prognosis in epithelial ovarian cancer. Int J Gynecol Cancer 20:1498–1505Google Scholar
  39. 39.
    Tan MJ, Teo Z, Sng MK, Zhu P, Tan NS (2012) Emerging roles of angiopoietin-like 4 in human cancer. Mol Cancer Res 10:677–688CrossRefGoogle Scholar
  40. 40.
    Santin AD, Hermonat PL, Ravaggi A, Cannon MJ, Pecorelli S, Parham GP (1999) Secretion of vascular endothelial growth factor in ovarian cancer. Eur J Gynaecol Oncol 20:177–181Google Scholar
  41. 41.
    Fagiani E, Christofori G (2013) Angiopoietins in angiogenesis. Cancer Lett 328:18–26CrossRefGoogle Scholar
  42. 42.
    Huang RL, Teo Z, Chong HC, Zhu P, Tan MJ, Tan CK, Lam CR, Sng MK, Leong DT, Tan SM et al (2011) ANGPTL4 modulates vascular junction integrity by integrin signaling and disruption of intercellular VE-cadherin and claudin-5 clusters. Blood 118:3990–4002CrossRefGoogle Scholar
  43. 43.
    Hakanpaa L, Sipila T, Leppanen VM, Gautam P, Nurmi H, Jacquemet G, Eklund L, Ivaska J, Alitalo K, Saharinen P (2015) Endothelial destabilization by angiopoietin-2 via integrin beta1 activation. Nat Commun 6:5962CrossRefGoogle Scholar
  44. 44.
    Roviezzo F, Tsigkos S, Kotanidou A, Bucci M, Brancaleone V, Cirino G, Papapetropoulos A (2005) Angiopoietin-2 causes inflammation in vivo by promoting vascular leakage. J Pharmacol Exp Ther 314:738–744CrossRefGoogle Scholar
  45. 45.
    Nagy JA, Herzberg KT, Dvorak JM, Dvorak HF (1993) Pathogenesis of malignant ascites formation: initiating events that lead to fluid accumulation. Cancer Res 53:2631–2643Google Scholar
  46. 46.
    Papadopoulos KP, Kelley RK, Tolcher AW, Razak AR, Van Loon K, Patnaik A, Bedard PL, Alfaro AA, Beeram M, Adriaens L et al (2016) A phase I first-in-human study of nesvacumab (REGN910), a fully human anti-angiopoietin-2 (Ang2) monoclonal antibody, in patients with advanced solid tumors. Clin Cancer Res 22:1348–1355CrossRefGoogle Scholar
  47. 47.
    Mazzieri R, Pucci F, Moi D, Zonari E, Ranghetti A, Berti A, Politi LS, Gentner B, Brown JL, Naldini L, de Palma M (2011) Targeting the ANG2/TIE2 axis inhibits tumor growth and metastasis by impairing angiogenesis and disabling rebounds of proangiogenic myeloid cells. Cancer Cell 19:512–526CrossRefGoogle Scholar
  48. 48.
    Liu N, Cui C, Sun Y, Zhang F, Wang S, Su G, Cai X (2017) Hydrogen peroxide promotes the expression of angiopoietin like 4 in RAW264.7 macrophages via MAPK pathways. Mol Med Rep 16:6128–6133CrossRefGoogle Scholar
  49. 49.
    Niu G, Carter WB (2007) Human epidermal growth factor receptor 2 regulates angiopoietin-2 expression in breast cancer via AKT and mitogen-activated protein kinase pathways. Cancer Res 67:1487–1493CrossRefGoogle Scholar
  50. 50.
    Tuppurainen L, Sallinen H, Karvonen A, Valkonen E, Laakso H, Liimatainen T, Hytonen E, Hamalainen K, Kosma VM, Anttila M et al (2017) Combined gene therapy using AdsVEGFR2 and AdsTie2 with chemotherapy reduces the growth of human ovarian cancer and formation of ascites in mice. Int J Gynecol Cancer 27:879–886CrossRefGoogle Scholar
  51. 51.
    Hamilton JR, Trejo J (2017) Challenges and opportunities in protease-activated receptor drug development. Annu Rev Pharmacol Toxicol 57:349–373CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Gregory D. Conway
    • 1
  • Marguerite S. Buzza
    • 1
  • Erik W. Martin
    • 1
    • 2
  • Nadire Duru
    • 1
  • Tierra A. Johnson
    • 1
  • Raymond J. Peroutka
    • 1
  • Nisha R. Pawar
    • 1
  • Toni M. Antalis
    • 1
    Email author
  1. 1.Center for Vascular and Inflammatory Diseases, Department of Physiology, and the University of Maryland Marlene and Stewart Greenebaum Comprehensive Cancer CenterUniversity of Maryland School of MedicineBaltimoreUSA
  2. 2.Laboratory of Molecular Biology and ImmunologyNational Institute on Aging, NIHBaltimoreUSA

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