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Gut microbiota-stimulated cathepsin K secretion mediates TLR4-dependent M2 macrophage polarization and promotes tumor metastasis in colorectal cancer

Cell Death & Differentiation (2019) | Download Citation


Metastasis is a complex process that requires the interaction between tumor cells and their microenvironment. As an important regulator of intestinal microenvironment, gut microbiota plays a significant role in the initiation and progression of colorectal cancer (CRC), but the underlying mechanisms remain elusive. In this study, a metastasis-related secretory protein cathepsin K (CTSK) was identified as a vital mediator between the imbalance of intestinal microbiota and CRC metastasis. We implanted MC38 cells into the cecal mesentry of antibiotic-treated mice with intragastrically administration of E. coli to mimic gut microbiota imbalance. The bigger primary tumors and more liver metastatic foci were detected in the E. coli group accompanied with high LPS secretion and CTSK overexpression compared with that in the control group. CTSK contributes to the aggressive phenotype of CRC cells both in vitro and in vivo. Silencing CTSK or administration of Odanacatib, a CTSK-specific inhibitor, totally abolished the CTSK-enhanced migration and motility of CRC cells. Interestingly, the tumor-secreted CTSK could bind to toll-like receptor 4 (TLR4) to stimulate the M2 polarization of tumor-associated macrophages (TAMs) via an mTOR-dependent pathway. Recombinant CTSK could neither stimulate CRC growth and metastasis, nor induce M2 macrophage polarization in TRL4−/− mice. Meanwhile, CTSK could stimulate the secretion of cytokines by M2 TAMs including IL10 and IL17, which, in turn, promote the invasion and metastasis of CRC cells through NFκB pathway. Clinically, overexpression of CTSK in human CRC tissues is always accompanied with high M2 TAMs in the stroma, and correlated with CRC metastasis and poor prognosis. Our current research identified CTSK as a mediator between the imbalance of gut microbiota and CRC metastasis. More importantly, we illustrated a CTSK-mediated-positive feedback loop between CRC cells and TAMs during metastasis, prompting CTSK as a novel predictive biomarker and feasible therapeutic target for CRC.

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  1. 1.

    De Rosa M, Pace U, Rega D, Costabile V, Duraturo F, Izzo P, et al. Genetics, diagnosis and management of colorectal cancer (Review). Oncol Rep. 2015;34:1087–96.

  2. 2.

    Joyce JA, Fearon DT. T cell exclusion, immune privilege, and the tumor microenvironment. Science. 2015;348:74–80.

  3. 3.

    Chung L, Orberg ET, Geis AL, Chan JL, Fu K, DeStefano Shields CE, et al. Bacteroides fragilis toxin coordinates a pro-carcinogenic inflammatory cascade via targeting of colonic epithelial cells. Cell Host Microbe. 2018;23:421.

  4. 4.

    Dejea CM, Fathi P, Craig JM, Boleij A, Taddese R, Geis AL, et al. Patients with familial adenomatous polyposis harbor colonic biofilms containing tumorigenic bacteria. Science. 2018;359:592–7.

  5. 5.

    Routy B, Le Chatelier E, Derosa L, Duong CPM, Alou MT, Daillere R, et al. Gut microbiome influences efficacy of PD-1-based immunotherapy against epithelial tumors. Science. 2018;359:91–7.

  6. 6.

    Singh N, Gurav A, Sivaprakasam S, Brady E, Padia R, Shi H, et al. Activation of Gpr109a, receptor for niacin and the commensal metabolite butyrate, suppresses colonic inflammation and carcinogenesis. Immunity. 2014;40:128–39.

  7. 7.

    Rooks MG, Garrett WS. Gut microbiota, metabolites and host immunity. Nat Rev Immunol. 2016;16:341–52.

  8. 8.

    Novinec M, Lenarcic B. Cathepsin K: a unique collagenolytic cysteine peptidase. Biol Chem. 2013;394:1163–79.

  9. 9.

    Aguda AH, Panwar P, Du X, Nguyen NT, Brayer GD, Bromme D. Structural basis of collagen fiber degradation by cathepsin K. Proc Natl Acad Sci USA. 2014;111:17474–9.

  10. 10.

    Leusink FK, Koudounarakis E, Frank MH, Koole R, van Diest PJ, Willems SM. Cathepsin K associates with lymph node metastasis and poor prognosis in oral squamous cell carcinoma. Bmc Cancer. 2018;18:385.

  11. 11.

    Duong LT, Wesolowski GA, Leung P, Oballa R, Pickarski M. Efficacy of a cathepsin K inhibitor in a preclinical model for prevention and treatment of breast cancer bone metastasis. Mol Cancer Ther. 2014;13:2898–909.

  12. 12.

    Xu H, Ma Y, Zhang Y, Pan Z, Lu Y, Liu P, et al. Identification of cathepsin K in the peritoneal metastasis of ovarian carcinoma using in-silico, gene expression analysis. J Cancer. 2016;7:722–9.

  13. 13.

    Verbovsek U, Motaln H, Rotter A, Atai NA, Gruden K, Van Noorden CJ, et al. Expression analysis of all protease genes reveals cathepsin K to be overexpressed in glioblastoma. PLoS ONE. 2014;9:e111819.

  14. 14.

    Christensen J, Shastri VP. Matrix-metalloproteinase-9 is cleaved and activated by cathepsin K. BMC Res Notes. 2015;8:322.

  15. 15.

    Kleer CG, Bloushtain-Qimron N, Chen YH, Carrasco D, Hu M, Yao J, et al. Epithelial and stromal cathepsin K and CXCL14 expression in breast tumor progression. Clin Cancer Res. 2008;14:5357–67.

  16. 16.

    Zhao L, Wang H, Liu C, Liu Y, Wang X, Wang S, et al. Promotion of colorectal cancer growth and metastasis by the LIM and SH3 domain protein 1. Gut. 2010;59:1226–35.

  17. 17.

    Cummins J, Cronin M, van Pijkeren JP, Gahan CG, Tangney M. Bacterial systems for gene delivery to systemic tumors. Methods Mol Biol. 2014;1141:201–9.

  18. 18.

    Hu J, Luo H, Wang J, Tang W, Lu J, Wu S, et al. Enteric dysbiosis-linked gut barrier disruption triggers early renal injury induced by chronic high salt feeding in mice. Exp Mol Med. 2017;49:e370.

  19. 19.

    Nimri L, Spivak O, Tal D, Schalling D, Peri I, Graeve L, et al. A recombinant fungal compound induces anti-proliferative and pro-apoptotic effects on colon cancer cells. Oncotarget. 2017;8:28854–64.

  20. 20.

    Zhang X, Goncalves R, Mosser DM. The isolation and characterization of murine macrophages. Curr Protoc Immunol. 2008; Chapter 14: Unit 14.1.

  21. 21.

    Liao Q, Li R, Zhou R, Pan Z, Xu L, Ding Y, et al. LIM kinase 1 interacts with myosin-9 and alpha-actinin-4 and promotes colorectal cancer progression. Br J Cancer. 2017;117:563–71.

  22. 22.

    Hirabara S, Kojima T, Takahashi N, Hanabayashi M, Ishiguro N. Hyaluronan inhibits TLR-4 dependent cathepsin K and matrix metalloproteinase 1 expression in human fibroblasts. Biochem Biophys Res Commun. 2013;430:519–22.

  23. 23.

    Caronni N, Savino B, Bonecchi R. Myeloid cells in cancer-related inflammation. Immunobiology. 2015;220:249–53.

  24. 24.

    Tilg H, Adolph TE, Gerner RR, Moschen AR. The intestinal microbiota in colorectal cancer. Cancer Cell. 2018;33:954–64.

  25. 25.

    Schulz MD, Atay C, Heringer J, Romrig FK, Schwitalla S, Aydin B, et al. High-fat-diet-mediated dysbiosis promotes intestinal carcinogenesis independently of obesity. Nature. 2014;514:508–12.

  26. 26.

    Jorgensen SF, Troseid M, Kummen M, Anmarkrud JA, Michelsen AE, Osnes LT, et al. Altered gut microbiota profile in common variable immunodeficiency associates with levels of lipopolysaccharide and markers of systemic immune activation. Mucosal Immunol. 2016;9:1455–65.

  27. 27.

    Cordes C, Bartling B, Simm A, Afar D, Lautenschlager C, Hansen G, et al. Simultaneous expression of Cathepsins B and K in pulmonary adenocarcinomas and squamous cell carcinomas predicts poor recurrence-free and overall survival. Lung Cancer. 2009;64:79–85.

  28. 28.

    Herroon MK, Rajagurubandara E, Rudy DL, Chalasani A, Hardaway AL, Podgorski I. Macrophage cathepsin K promotes prostate tumor progression in bone. Oncogene. 2013;32:1580–93.

  29. 29.

    Kos J, Sekirnik A, Premzl A, Zavasnik Bergant V, Langerholc T, Turk B, et al. Carboxypeptidases cathepsins X and B display distinct protein profile in human cells and tissues. Exp Cell Res. 2005;306:103–13.

  30. 30.

    Guiet R, Van Goethem E, Cougoule C, Balor S, Valette A, Al Saati T, et al. The process of macrophage migration promotes matrix metalloproteinase-independent invasion by tumor cells. J Immunol. 2011;187:3806–14.

  31. 31.

    Tan SY, Fan Y, Luo HS, Shen ZX, Guo Y, Zhao LJ. Prognostic significance of cell infiltrations of immunosurveillance in colorectal cancer. World J Gastroenterol. 2005;11:1210–4.

  32. 32.

    Cui YL, Li HK, Zhou HY, Zhang T, Li Q. Correlations of tumor-associated macrophage subtypes with liver metastases of colorectal cancer. Asian Pac J Cancer Prev. 2013;14:1003–7.

  33. 33.

    Duvel K, Yecies JL, Menon S, Raman P, Lipovsky AI, Souza AL, et al. Activation of a metabolic gene regulatory network downstream of mTOR complex 1. Mol Cell. 2010;39:171–83.

  34. 34.

    Salmiheimo AN, Mustonen HK, Vainionpaa SA, Shen Z, Kemppainen EA, Seppanen HE, et al. Increasing the inflammatory competence of macrophages with IL-6 or with combination of IL-4 and LPS restrains the invasiveness of pancreatic cancer cells. J Cancer. 2016;7:42–9.

  35. 35.

    Fan Y, Liu B. Expression of toll-like receptors in the mucosa of patients with ulcerative colitis. Exp Ther Med. 2015;9:1455–9.

  36. 36.

    Watanabe T, Takahashi N, Hirabara S, Ishiguro N, Kojima T. Hyaluronan inhibits Tlr-4-dependent RANKL expression in human rheumatoid arthritis synovial fibroblasts. PLoS ONE. 2016;11:e0153142.

  37. 37.

    Donners MM, Bai L, Lutgens SP, Wijnands E, Johnson J, Schurgers LJ, et al. Cathepsin K deficiency prevents the aggravated vascular remodeling response to flow cessation in ApoE-/- mice. PLoS ONE. 2016;11:e0162595.

  38. 38.

    Sun Y, Ishibashi M, Seimon T, Lee M, Sharma SM, Fitzgerald KA, et al. Free cholesterol accumulation in macrophage membranes activates Toll-like receptors and p38 mitogen-activated protein kinase and induces cathepsin K. Circ Res. 2009;104:455–65.

  39. 39.

    Popena I, Abols A, Saulite L, Pleiko K, Zandberga E, Jekabsons K, et al. Effect of colorectal cancer-derived extracellular vesicles on the immunophenotype and cytokine secretion profile of monocytes and macrophages. Cell Commun Signal. 2018;16:17.

  40. 40.

    Gaffen SL. Structure and signalling in the IL-17 receptor family. Nat Rev Immunol. 2009;9:556–67.

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This work was supported by the National Natural Science Foundation of China (Nos. 81572813, 81773082, 81702903, 81872423), Guangdong Natural Science Foundation (2017A030310038, 2018B030311036) and Fork Ying Tung Education Foundation (161035).

Author information

Author notes

  1. These authors contributed equally: Rui Li, Rui Zhou, Hui Wang


  1. Department of Pathology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, China

    • Rui Li
    • , Rui Zhou
    • , Yanqing Ding
    •  & Liang Zhao
  2. Department of Pathology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, Guangdong, China

    • Rui Li
    • , Rui Zhou
    • , Wanqi Zhan
    • , Lijun Xu
    • , Yanqing Ding
    •  & Liang Zhao
  3. Department of Medical Oncology, Affiliated Tumor Hospital of Guangzhou Medical University, Guangzhou, China

    • Hui Wang
    •  & Weidong Li
  4. Second Department of Hepatobiliary Surgery, Zhujiang Hospital, Southern Medical University, Guangzhou, China

    • Mingxin Pan
  5. Department of General Surgery, Guangdong General Hospital, Guangdong Academy of Medical Science, Guangzhou, China

    • Xueqing Yao
  6. Gastrointestinal Surgical Center, First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China

    • Shibin Yang


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LZ led study design and prepared the manuscript; RL, RZ, and HW carried out the experiments; W-DL, L-JX and M-XP performed statistical analysis; X-QY, W-QZ and S-BY assisted in tissue sample collection and clinical analysis; Y-QD performed data analysis and interpretation.

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The authors declare that they have no conflict of interest.

Corresponding authors

Correspondence to Yanqing Ding or Liang Zhao.

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