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Targeting an autocrine IL-6–SPINK1 signaling axis to suppress metastatic spread in ovarian clear cell carcinoma


A major clinical challenge of ovarian cancer is the development of malignant ascites accompanied by widespread peritoneal metastasis. In ovarian clear cell carcinoma (OCCC), a challenging subtype of ovarian cancer, this problem is compounded by near-universal primary chemoresistance; patients with advanced stage OCCC thus lack effective therapies and face extremely poor survival rates. Here we show that tumor-cell-expressed serine protease inhibitor Kazal type 1 (SPINK1) is a key driver of OCCC progression and metastasis. Using cell culture models of human OCCC, we find that shRNA silencing of SPINK1 sensitizes tumor cells to anoikis and inhibits proliferation. Knockdown of SPINK1 in OCCC cells also profoundly suppresses peritoneal metastasis in mouse implantation models of human OCCC. We next identify a novel autocrine signaling axis in OCCC cells whereby tumor-cell-produced interleukin-6 (IL-6) regulates SPINK1 expression to stimulate a common protumorigenic gene expression pattern leading to anoikis resistance and proliferation of OCCC cells. We further demonstrate that this signaling pathway can be successfully interrupted with the IL-6Rα inhibitor tocilizumab, sensitizing cells to anoikis in vitro and reducing metastasis in vivo. These results suggest that clinical trials of IL-6 pathway inhibitors in OCCC may be warranted, and that SPINK1 might offer a candidate predictive biomarker in this population.

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Fig. 1: SPINK1 knockdown sensitizes OCCC tumor cells to anoikis and inhibits proliferation.
Fig. 2: SPINK1 knockdown in OCCC tumor cells reduces tumor burden and inhibits peritoneal metastasis.
Fig. 3: The IL-6 signaling pathway drives SPINK1 expression in OCCC.
Fig. 4: IL-6 increases tumor cell survival and tumor cell proliferation through SPINK1 expression.
Fig. 5: Common gene expression patterns with knockdown of SPINK1 and IL-6.
Fig. 6: Tocilizumab targets IL-6 signaling to reduce SPINK1 expression, tumor cell survival, ascites accumulation and metastasis.


  1. 1.

    Howlader N, Noone AM, Krapcho M, Miller D, Brest A, Yu M, et al. SEER cancer statistics review, 1975–2016. Bethesda, MD: National Cancer Institute; 2017.

  2. 2.

    Lengyel E. Ovarian Cancer Development and Metastasis. Am J Pathol. 2010;177:1053–64.

    PubMed  PubMed Central  Google Scholar 

  3. 3.

    Mitra AK. Ovarian cancer metastasis: a unique mechanism of dissemination. In: Xu K (editor). Tumor metastasis. Rijeka: InTech; 2016.

  4. 4.

    du Bois A, Lück H-J, Meier W, Adams H-P, Möbus V, Costa S, et al. A randomized clinical trial of cisplatin/paclitaxel versus carboplatin/paclitaxel as first-line treatment of ovarian cancer. J Natl Cancer Inst. 2003;95:1320–9.

    PubMed  Google Scholar 

  5. 5.

    Sugiyama T, Kamura T, Kigawa J, Terakawa N, Kikuchi Y, Kita T, et al. Clinical characteristics of clear cell carcinoma of the ovary: a distinct histologic type with poor prognosis and resistance to platinum-based chemotherapy. Cancer. 2000;88:2584–9.

    CAS  PubMed  Google Scholar 

  6. 6.

    Takano M, Kikuchi Y, Yaegashi N. Clear cell carcinoma of the ovary: a retrospective multicentre experience of 254 patients with complete surgical staging. Br J Cancer. 2006;94:1369–74.

  7. 7.

    del Carmen MG, Birrer M, Schorge JO. Clear cell carcinoma of the ovary: a review of the literature. Gynecologic Oncol. 2012;126:481–90.

    Google Scholar 

  8. 8.

    Chan JK, Teoh D, Hu JM, Shin JY, Osann K, Kapp DS. Do clear cell ovarian carcinomas have poorer prognosis compared to other epithelial cell types? A study of 1411 clear cell ovarian cancers. Gynecologic Oncol. 2008;109:370–6.

    Google Scholar 

  9. 9.

    Ahmed N, Stenvers KL. Getting to know ovarian cancer ascites: opportunities for targeted therapy-based translational research. Front Oncol. 2013;3:256.

    PubMed  PubMed Central  Google Scholar 

  10. 10.

    Simpson CD, Anyiwe K, Schimmer AD. Anoikis resistance and tumor metastasis. Cancer Lett. 2008;272:177–85.

    CAS  PubMed  Google Scholar 

  11. 11.

    Mehner C, Oberg AL, Kalli KR, Nassar A, Hockla A, Pendlebury D, et al. Serine protease inhibitor Kazal type 1 (SPINK1) drives proliferation and anoikis resistance in a subset of ovarian cancers. Oncotarget. 2015;6:35737–54.

    PubMed  PubMed Central  Google Scholar 

  12. 12.

    Rinderknecht H. Activation of pancreatic zymogens. Normal activation, premature intrapancreatic activation, protective mechanisms against inappropriate activation. Dig Dis Sci (Rev). 1986;31:314–21.

    CAS  Google Scholar 

  13. 13.

    Itkonen O, Stenman UH. TATI as a biomarker. Clin Chim Acta; Int J Clin Chem. 2014;431:260–9.

    CAS  Google Scholar 

  14. 14.

    Rasanen K, Itkonen O, Koistinen H, Stenman UH. Emerging roles of SPINK1 in cancer. Clin Chem. 2016;62:449–57.

    PubMed  Google Scholar 

  15. 15.

    Stenman UH. Role of the tumor-associated trypsin inhibitor SPINK1 in cancer development. Asian J Androl. 2011;13:628–9.

    PubMed  PubMed Central  Google Scholar 

  16. 16.

    Hunter CA, Jones SA. IL-6 as a keystone cytokine in health and disease. Nat Immunol (Rev). 2015;16:448–57.

    CAS  Google Scholar 

  17. 17.

    Taniguchi K, Karin M. IL-6 and related cytokines as the critical lynchpins between inflammation and cancer. Semin Immunol. 2014;26:54–74.

    CAS  PubMed  Google Scholar 

  18. 18.

    Yasuda T, Ogawa M, Murata A, Oka Y, Uda K, Mori T. Response to IL-6 stimulation of human hepatoblastoma cells: production of pancreatic secretory trypsin inhibitor. Biol Chem Hoppe Seyler. 1990;371(Suppl):95–100.

    CAS  PubMed  Google Scholar 

  19. 19.

    Yasuda T, Ogawa M, Murata A, Ohmachi Y, Yasuda T, Mori T, et al. Identification of the IL-6-responsive element in an acute-phase-responsive human pancreatic secretory trypsin inhibitor-encoding gene. Gene. 1993;131:275–80.

    CAS  PubMed  Google Scholar 

  20. 20.

    Rasanen K, Lehtinen E, Nokelainen K, Kuopio T, Hautala L, Itkonen O, et al. Interleukin-6 increases expression of serine protease inhibitor Kazal type 1 through STAT3 in colorectal adenocarcinoma. Mol Carcinog. 2016;55:2010–23.

  21. 21.

    Watson JM, Sensintaffar JL, Berek JS, Martinez-Maza O. Constitutive production of interleukin 6 by ovarian cancer cell lines and by primary ovarian tumor cultures. Cancer Res. 1990;50:6959–65.

    CAS  PubMed  Google Scholar 

  22. 22.

    Nilsson MB, Langley RR, Fidler IJ. Interleukin-6, secreted by human ovarian carcinoma cells, is a potent proangiogenic cytokine. Cancer Res. 2005;65:10794–800.

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Anglesio MS, George J, Kulbe H, Friedlander M, Rischin D, Lemech C, et al. IL6-STAT3-HIF signaling and therapeutic response to the angiogenesis inhibitor sunitinib in ovarian clear cell cancer. Clin Cancer Res. 2011;17:2538–48.

    CAS  PubMed  Google Scholar 

  24. 24.

    Chandler RL, Damrauer JS, Raab JR, Schisler JC, Wilkerson MD, Didion JP, et al. Coexistent ARID1A–PIK3CA mutations promote ovarian clear-cell tumorigenesis through pro-tumorigenic inflammatory cytokine signalling. Nat Commun. 2015;6:6118.

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Kawabata A, Yanaihara N, Nagata C, Saito M, Noguchi D, Takenaka M, et al. Prognostic impact of interleukin-6 expression in stage I ovarian clear cell carcinoma. Gynecologic Oncol. 2017;146:609–14.

    CAS  Google Scholar 

  26. 26.

    Yanaihara N, Hirata Y, Yamaguchi N, Noguchi Y, Saito M, Nagata C, et al. Antitumor effects of interleukin-6 (IL-6)/interleukin-6 receptor (IL-6R) signaling pathway inhibition in clear cell carcinoma of the ovary. Mol Carcinog. 2016;55:832–41.

    CAS  PubMed  Google Scholar 

  27. 27.

    Stany MP, Vathipadiekal V, Ozbun L, Stone RL, Mok SC, Xue H, et al. Identification of novel therapeutic targets in microdissected clear cell ovarian cancers. PLoS ONE. 2011;6:e21121.

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Yuniati L, Scheijen B, van der Meer LT, van Leeuwen FN. Tumor suppressors BTG1 and BTG2: Beyond growth control. J Cell Physiol. 2019;234:5379–89.

    CAS  PubMed  Google Scholar 

  29. 29.

    Hirano G, Izumi H, Yasuniwa Y, Shimajiri S, Ke-Yong W, Sasagiri Y, et al. Involvement of riboflavin kinase expression in cellular sensitivity against cisplatin. Int J Oncol. 2011;38:893–902.

    CAS  PubMed  Google Scholar 

  30. 30.

    Cepeda D, Ng HF, Sharifi HR, Mahmoudi S, Cerrato VS, Fredlund E, et al. CDK-mediated activation of the SCF(FBXO) (28) ubiquitin ligase promotes MYC-driven transcription and tumourigenesis and predicts poor survival in breast cancer. EMBO Mol Med. 2013;5:1067–86.

    PubMed  Google Scholar 

  31. 31.

    Marzec K, Burgess A. The oncogenic functions of MASTL kinase. Front Cell Dev Biol. 2018;6:162

    PubMed  PubMed Central  Google Scholar 

  32. 32.

    Stenman U-H. Tumor-associated trypsin inhibitor. Clin Chem. 2002;48:1206–9.

    CAS  PubMed  Google Scholar 

  33. 33.

    Tomlins SA, Rhodes DR, Yu J, Varambally S, Mehra R, Perner S, et al. The role of SPINK1 in ETS rearrangement-negative prostate cancers. Cancer Cell. 2008;13:519–28.

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Soon WW, Miller LD, Black MA, Dalmasso C, Chan XB, Pang B, et al. Combined genomic and phenotype screening reveals secretory factor SPINK1 as an invasion and survival factor associated with patient prognosis in breast cancer. EMBO Mol Med. 2011;3:451–64.

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Halila H, Huhtala ML, Haglund C, Nordling S, Stenman UH. Tumour-associated trypsin inhibitor (TATI) in human ovarian cyst fluid. Comparison with CA 125 and CEA. Br J Cancer. 1987;56:153–6.

    CAS  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Huhtala ML, Pesonen K, Kalkkinen N, Stenman UH. Purification and characterization of a tumor-associated trypsin inhibitor from the urine of a patient with ovarian cancer. J Biol Chem. 1982;257:13713–6.

    CAS  PubMed  Google Scholar 

  37. 37.

    Paju A, Vartiainen J, Haglund C, Itkonen O, von Boguslawski K, Leminen A, et al. Expression of trypsinogen-1, trypsinogen-2, and tumor-associated trypsin inhibitor in ovarian cancer: prognostic study on tissue and serum. Clin Cancer Res. 2004;10:4761–8.

    CAS  PubMed  Google Scholar 

  38. 38.

    Ateeq B, Tomlins SA, Laxman B, Asangani IA, Cao Q, Cao X, et al. Therapeutic targeting of SPINK1-positive prostate cancer. Sci Transl Med. 2011;3:72ra17.

    PubMed  PubMed Central  Google Scholar 

  39. 39.

    Lamontagne J, Pinkerton M, Block TM, Lu X, Hepatitis B, Hepatitis C. Virus replication upregulates serine protease inhibitor Kazal, resulting in cellular resistance to serine protease-dependent apoptosis. J Virol. 2010;84:907–17.

    CAS  PubMed  Google Scholar 

  40. 40.

    Lu X, Lamontagne J, Lu F, Block TM. Tumor-associated protein SPIK/TATI suppresses serine protease dependent cell apoptosis. Apoptosis: Int J Program Cell Death. 2008;13:483–94.

    CAS  Google Scholar 

  41. 41.

    Chen F, Long Q, Fu D, Zhu D, Ji Y, Han L, et al. Targeting SPINK1 in the damaged tumour microenvironment alleviates therapeutic resistance. Nat Commun. 2018;9:4315.

    PubMed  PubMed Central  Google Scholar 

  42. 42.

    Wiegand KC, Shah SP, Al-Agha OM, Zhao Y, Tse K, Zeng T, et al. ARID1A mutations in endometriosis-associated ovarian carcinomas. N Engl J Med. 2010;363:1532–43.

    CAS  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Sugiyama T, Yakushiji M, Nishida T. Irinotecan (CPT-11) combined with cisplatin in patients with refractory or recurrent ovarian cancer. Cancer Lett. 1998;128:211–8.

  44. 44.

    Anglesio MS, Wiegand KC, Melnyk N, Chow C, Salamanca C, Prentice LM, et al. Type-specific cell line models for type-specific ovarian cancer research. PLoS ONE. 2013;8:e72162.

    CAS  PubMed  PubMed Central  Google Scholar 

  45. 45.

    Dijkgraaf EM, Santegoets SJ, Reyners AK, Goedemans R, Wouters MC, Kenter GG, et al. A phase I trial combining carboplatin/doxorubicin with tocilizumab, an anti-IL-6R monoclonal antibody, and interferon-alpha2b in patients with recurrent epithelial ovarian cancer. Ann Oncol: Off J Eur Soc Med Oncol. 2015;26:2141–9.

    CAS  Google Scholar 

  46. 46.

    Coward J, Kulbe H, Chakravarty P, Leader D, Vassileva V, Leinster DA, et al. Interleukin-6 as a therapeutic target in human ovarian cancer. Clin Cancer Res. 2011;17:6083–96.

    CAS  PubMed  PubMed Central  Google Scholar 

  47. 47.

    Angevin E, Tabernero J, Elez E, Cohen SJ, Bahleda R, van Laethem JL, et al. A phase I/II, multiple-dose, dose-escalation study of siltuximab, an anti-interleukin-6 monoclonal antibody, in patients with advanced solid tumors. Clin Cancer Res. 2014;20:2192–204.

    CAS  PubMed  Google Scholar 

  48. 48.

    Pectasides D, Pectasides E, Psyrri A, Economopoulos T. Treatment issues in clear cell carcinoma of the ovary: a different entity? oncologist. 2006;11:1089–94.

    PubMed  Google Scholar 

  49. 49.

    Kipps E, Tan DS, Kaye SB. Meeting the challenge of ascites in ovarian cancer: new avenues for therapy and research. Nat Rev Cancer. 2013;13:273–82.

    CAS  PubMed  PubMed Central  Google Scholar 

  50. 50.

    Al-Quteimat OM, Al-Badaineh MA. Intraperitoneal chemotherapy: rationale, applications, and limitations. J Oncol Pharm Pract: Off Publ Int Soc Oncol Pharm Pract. 2014;20:369–80.

    CAS  Google Scholar 

  51. 51.

    Kereszturi E, Kiraly O, Sahin-Toth M. Minigene analysis of intronic variants in common SPINK1 haplotypes associated with chronic pancreatitis. Gut. 2009;58:545–9.

    CAS  PubMed  Google Scholar 

  52. 52.

    Portolano N, Watson PJ, Fairall L, Millard CJ, Milano CP, Song Y, et al. Recombinant protein expression for structural biology in HEK 293F suspension cells: a novel and accessible approach. J Vis Exp. 2014;92:e51897.

  53. 53.

    Cichon MA, Nelson CM, Radisky DC. Regulation of epithelial-mesenchymal transition in breast cancer cells by cell contact and adhesion. Cancer Inf. 2015;14:1–13.

    CAS  Google Scholar 

  54. 54.

    Ma H, Hockla A, Mehner C, Coban M, Papo N, Radisky DC, et al. PRSS3/Mesotrypsin and kallikrein-related peptidase 5 are associated with poor prognosis and contribute to tumor cell invasion and growth in lung adenocarcinoma. Sci Rep. 2019;9:1844.

    PubMed  PubMed Central  Google Scholar 

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We acknowledge Brandy Edenfield of the Mayo Clinic Department of Cancer Biology histology shared resource for excellent technical support, and Dr. Laura Lewis-Tuffin for excellent technical support at the Mayo Clinic Flow Cytometry Core.

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CM and ESR designed and created this study, analyzed and interpreted the data, and drafted the manuscript. CM, AH, and MC performed in vitro assays. SJW provided training and expertise for ultra-low attachment experiments. CM and EM performed the in vivo studies. DCR performed microarray analysis and contributed to the drafting of the manuscript. All authors approved the final manuscript.

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Correspondence to Evette S. Radisky.

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Mehner, C., Miller, E., Hockla, A. et al. Targeting an autocrine IL-6–SPINK1 signaling axis to suppress metastatic spread in ovarian clear cell carcinoma. Oncogene 39, 6606–6618 (2020).

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