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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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.
Lengyel E. Ovarian Cancer Development and Metastasis. Am J Pathol. 2010;177:1053–64.
Mitra AK. Ovarian cancer metastasis: a unique mechanism of dissemination. In: Xu K (editor). Tumor metastasis. Rijeka: InTech; 2016.
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.
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.
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.
del Carmen MG, Birrer M, Schorge JO. Clear cell carcinoma of the ovary: a review of the literature. Gynecologic Oncol. 2012;126:481–90.
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.
Ahmed N, Stenvers KL. Getting to know ovarian cancer ascites: opportunities for targeted therapy-based translational research. Front Oncol. 2013;3:256.
Simpson CD, Anyiwe K, Schimmer AD. Anoikis resistance and tumor metastasis. Cancer Lett. 2008;272:177–85.
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.
Rinderknecht H. Activation of pancreatic zymogens. Normal activation, premature intrapancreatic activation, protective mechanisms against inappropriate activation. Dig Dis Sci (Rev). 1986;31:314–21.
Itkonen O, Stenman UH. TATI as a biomarker. Clin Chim Acta; Int J Clin Chem. 2014;431:260–9.
Rasanen K, Itkonen O, Koistinen H, Stenman UH. Emerging roles of SPINK1 in cancer. Clin Chem. 2016;62:449–57.
Stenman UH. Role of the tumor-associated trypsin inhibitor SPINK1 in cancer development. Asian J Androl. 2011;13:628–9.
Hunter CA, Jones SA. IL-6 as a keystone cytokine in health and disease. Nat Immunol (Rev). 2015;16:448–57.
Taniguchi K, Karin M. IL-6 and related cytokines as the critical lynchpins between inflammation and cancer. Semin Immunol. 2014;26:54–74.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Marzec K, Burgess A. The oncogenic functions of MASTL kinase. Front Cell Dev Biol. 2018;6:162
Stenman U-H. Tumor-associated trypsin inhibitor. Clin Chem. 2002;48:1206–9.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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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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). https://doi.org/10.1038/s41388-020-01451-4