Toll-Like Receptor 4 and Matrix Metalloproteases 11 and 13 as Predictors of Tumor Recurrence and Survival in Stage II Colorectal Cancer

  • Noemi Eiro
  • Juan Francisco Carrión
  • Sandra Cid
  • Alejandro Andicoechea
  • José Luis García-Muñiz
  • Luis O. González
  • Francisco J. VizosoEmail author
Original Article


Current clinical-pathologic stratification factors do not allow clear identification of high-risk stage II colorectal cancer (CRC) patients. Therefore, the identification of additional prognostic markers is desirable. Toll-like receptor (TLR)-4 is activated during tumorigenesis and matrix metalloproteases (MMPs) are involved in invasion and metastasis. We aimed to evaluate the expression and clinical relevance of TLR4, MMP11 and MMP13 for patients with stage II CRC. Immunohistochemistry was used to study the expression of TLR4, MMP11 and MMP13 in 96 patients with stage II CRC. We measured the global expression and the expression by different cell types (tumor cells, cancer-associated fibroblasts (CAFs) and mononuclear inflammatory cells (MICs)). The potential relationship between expressions of factors and different prognostic variables were evaluated. Our results show significant relationships between either TLR4 expression by tumor cells and MMP11 expression by CAFs and high risk of tumor recurrence. In addition, the concurrence of age ≥ 75 years and the non-expression of MMP11 by CAFs identify a subgroup of patients with a good prognosis. Our results show that TLR4 expression by tumor cells and MMP11 expression by CAFs may to improve the identification of patients with stage II CRC with a high-risk of relapse.


Prognostic factor Stage II colorectal cancer Survival TLR4 MMP11 MMP13 


Author Contributions

NE, JFC and FV designed the study, analyzed and interpreted data. NE, JFC, SC, LOG, JLGM and AA collected data, carried out experiments, analyzed data and generated the figures. All authors were involved in writing the paper and had final approval of the submitted and published version. FV accepts full responsibility for the work and/or the conduct of the study, had access to the data, and oversaw the decision to publish.


This work was supported by grant from the Consejería de Economía y Empleo del Principado de Asturias (GRUPIN14–116) S.C. was recipient of a predoctoral fellowship financed by the Gobierno del Principado de Asturias “Severo Ochoa” PhD Program (BP14–128).

Compliance with Ethical Standards

Ethical Approval

The study is adhered to National regulations, and was approved by the Fundación Hospital de Jove Ethics and Investigation Committee.

Conflict of Interest

The authors declare that they have no conflicts of interest.


  1. 1.
    Tenesa A, Dunlop MG (2009) New insights into the aetiology of colorectal cancer from genome-wide association studies. Nat Rev Genet 10(6):353–358CrossRefGoogle Scholar
  2. 2.
    Jemal A, Siegel R, Xu J, Ward E (2010) Cancer statistics, 2010. CA Cancer J Clin 60(5):277–300CrossRefGoogle Scholar
  3. 3.
    Sobin LH, Gospodarowicz MK, Wittekind C (2011) TNM classification of malignant tumours. WileyGoogle Scholar
  4. 4.
    Bosman FT, Carneiro F, Hruban RH, Theise ND (2010) WHO classification of tumours of the digestive system. vol Ed. 4. World Health Organization,Google Scholar
  5. 5.
    Jessup JM, McGinnis LS, Steele GD Jr, Menck HR, Winchester DP (1996) The National Cancer Data Base. Report on colon cancer. Cancer 78(4):918–926CrossRefGoogle Scholar
  6. 6.
    Edge SB, Compton CC (2010) The American joint committee on Cancer: the 7th edition of the AJCC cancer staging manual and the future of TNM. Ann Surg Oncol 17(6):1471–1474CrossRefGoogle Scholar
  7. 7.
    O'Connell JB, Maggard MA, Ko CY (2004) Colon cancer survival rates with the new American joint committee on Cancer sixth edition staging. J Natl Cancer Inst 96(19):1420–1425CrossRefGoogle Scholar
  8. 8.
    La Torre M, Lorenzon L, Pilozzi E, Barucca V, Cavallini M, Ziparo V, Ferri M (2012) Number of harvested lymph nodes is the main prognostic factor in stage IIa colorectal cancer patients. J Surg Oncol 106(4):469–474CrossRefGoogle Scholar
  9. 9.
    Santos C, Lopez-Doriga A, Navarro M, Mateo J, Biondo S, Martinez Villacampa M, Soler G, Sanjuan X, Paules MJ, Laquente B, Guino E, Kreisler E, Frago R, Germa JR, Moreno V, Salazar R (2013) Clinicopathological risk factors of stage II colon cancer: results of a prospective study. Color Dis 15(4):414–422CrossRefGoogle Scholar
  10. 10.
    Hari DM, Leung AM, Lee JH, Sim MS, Vuong B, Chiu CG, Bilchik AJ (2013) AJCC Cancer staging manual 7th edition criteria for colon cancer: do the complex modifications improve prognostic assessment? J Am Coll Surg 217(2):181–190CrossRefGoogle Scholar
  11. 11.
    Fang SH, Efron JE, Berho ME, Wexner SD (2014) Dilemma of stage II colon cancer and decision making for adjuvant chemotherapy. J Am Coll Surg 219(5):1056–1069CrossRefGoogle Scholar
  12. 12.
    Compton C, Fenoglio-Preiser CM, Pettigrew N, Fielding LP (2000) American joint committee on Cancer prognostic factors consensus conference: colorectal working group. Cancer 88(7):1739–1757CrossRefGoogle Scholar
  13. 13.
    Akagi Y, Shirouzu K, Kinugasa T (2013) Extramural extension as indicator for postoperative adjuvant chemotherapy in stage IIA (pT3N0) colon cancer. J Surg Oncol 108(6):358–363CrossRefGoogle Scholar
  14. 14.
    Stocchi L, Fazio VW, Lavery I, Hammel J (2011) Individual surgeon, pathologist, and other factors affecting lymph node harvest in stage II colon carcinoma. Is a minimum of 12 examined lymph nodes sufficient? Ann Surg Oncol 18(2):405–412CrossRefGoogle Scholar
  15. 15.
    Harris EI, Lewin DN, Wang HL, Lauwers GY, Srivastava A, Shyr Y, Shakhtour B, Revetta F, Washington MK (2008) Lymphovascular invasion in colorectal cancer: an interobserver variability study. Am J Surg Pathol 32(12):1816–1821CrossRefGoogle Scholar
  16. 16.
    Rakoff-Nahoum S, Medzhitov R (2009) Toll-like receptors and cancer. Nat Rev Cancer 9(1):57–63CrossRefGoogle Scholar
  17. 17.
    Mantovani A, Allavena P, Sica A, Balkwill F (2008) Cancer-related inflammation. Nature 454(7203):436–444CrossRefGoogle Scholar
  18. 18.
    Castro FA, Forsti A, Buch S, Kalthoff H, Krauss C, Bauer M, Egberts J, Schniewind B, Broering DC, Schreiber S, Schmitt M, Hampe J, Hemminki K, Schafmayer C (2011) TLR-3 polymorphism is an independent prognostic marker for stage II colorectal cancer. Eur J Cancer 47(8):1203–1210CrossRefGoogle Scholar
  19. 19.
    O'Leary DP, Bhatt L, Woolley JF, Gough DR, Wang JH, Cotter TG, Redmond HP (2012) TLR-4 signalling accelerates colon cancer cell adhesion via NF-kappaB mediated transcriptional up-regulation of Nox-1. PLoS One 7(10):e44176CrossRefGoogle Scholar
  20. 20.
    Grimm M, Kim M, Rosenwald A, Heemann U, Germer CT, Waaga-Gasser AM, Gasser M (2010) Toll-like receptor (TLR) 7 and TLR8 expression on CD133+ cells in colorectal cancer points to a specific role for inflammation-induced TLRs in tumourigenesis and tumour progression. Eur J Cancer 46(15):2849–2857CrossRefGoogle Scholar
  21. 21.
    Rayburn ER, Wang W, Zhang R, Wang H (2007) Experimental therapy for colon cancer: anti-cancer effects of TLR9 agonism, combination with other therapeutic modalities, and dependence upon p53. Int J Oncol 30(6):1511–1519Google Scholar
  22. 22.
    Eiro N, Gonzalez L, Gonzalez LO, Fernandez-Garcia B, Andicoechea A, Barbon E, Garcia-Muniz JL, Vizoso FJ (2013) Toll-like receptor-4 expression by stromal fibroblasts is associated with poor prognosis in colorectal cancer. J Immunother 36(6):342–349CrossRefGoogle Scholar
  23. 23.
    Nelson AR, Fingleton B, Rothenberg ML, Matrisian LM (2000) Matrix metalloproteinases: biologic activity and clinical implications. J Clin Oncol 18(5):1135–1149CrossRefGoogle Scholar
  24. 24.
    Manes S, Llorente M, Lacalle RA, Gomez-Mouton C, Kremer L, Mira E, Martinez AC (1999) The matrix metalloproteinase-9 regulates the insulin-like growth factor-triggered autocrine response in DU-145 carcinoma cells. J Biol Chem 274(11):6935–6945CrossRefGoogle Scholar
  25. 25.
    Noe V, Fingleton B, Jacobs K, Crawford HC, Vermeulen S, Steelant W, Bruyneel E, Matrisian LM, Mareel M (2001) Release of an invasion promoter E-cadherin fragment by matrilysin and stromelysin-1. J Cell Sci 114(Pt 1):111–118Google Scholar
  26. 26.
    Egeblad M, Werb Z (2002) New functions for the matrix metalloproteinases in cancer progression. Nat Rev Cancer 2(3):161–174CrossRefGoogle Scholar
  27. 27.
    Turk V, Kos J, Turk B (2004) Cysteine cathepsins (proteases)--on the main stage of cancer? Cancer Cell 5(5):409–410CrossRefGoogle Scholar
  28. 28.
    Fingleton B, Vargo-Gogola T, Crawford HC, Matrisian LM (2001) Matrilysin [MMP-7] expression selects for cells with reduced sensitivity to apoptosis. Neoplasia 3(6):459–468CrossRefGoogle Scholar
  29. 29.
    Chen R, Cui J, Xu C, Xue T, Guo K, Gao D, Liu Y, Ye S, Ren Z (2012) The significance of MMP-9 over MMP-2 in HCC invasiveness and recurrence of hepatocellular carcinoma after curative resection. Ann Surg Oncol 19(Suppl 3):S375–S384CrossRefGoogle Scholar
  30. 30.
    Koskensalo S, Louhimo J, Nordling S, Hagstrom J, Haglund C (2011) MMP-7 as a prognostic marker in colorectal cancer. Tumour Biol 32(2):259–264CrossRefGoogle Scholar
  31. 31.
    Escaff S, Fernandez JM, Gonzalez LO, Suarez A, Gonzalez-Reyes S, Gonzalez JM, Vizoso FJ (2010) Study of matrix metalloproteinases and their inhibitors in prostate cancer. Br J Cancer 102(5):922–929CrossRefGoogle Scholar
  32. 32.
    Gonzalez LO, Pidal I, Junquera S, Corte MD, Vazquez J, Rodriguez JC, Lamelas ML, Merino AM, Garcia-Muniz JL, Vizoso FJ (2007) Overexpression of matrix metalloproteinases and their inhibitors in mononuclear inflammatory cells in breast cancer correlates with metastasis-relapse. Br J Cancer 97(7):957–963CrossRefGoogle Scholar
  33. 33.
    Pesta M, Topolcan O, Holubec L Jr, Rupert K, Cerna M, Holubec LS, Treska V, Finek J, Cerny R (2007) Clinicopathological assessment and quantitative estimation of the matrix metalloproteinases MMP-2 and MMP-7 and the inhibitors TIMP-1 and TIMP-2 in colorectal carcinoma tissue samples. Anticancer Res 27(4A):1863–1867Google Scholar
  34. 34.
    Asano T, Tada M, Cheng S, Takemoto N, Kuramae T, Abe M, Takahashi O, Miyamoto M, Hamada J, Moriuchi T, Kondo S (2008) Prognostic values of matrix metalloproteinase family expression in human colorectal carcinoma. J Surg Res 146(1):32–42CrossRefGoogle Scholar
  35. 35.
    Murray GI, Duncan ME, O'Neil P, Melvin WT, Fothergill JE (1996) Matrix metalloproteinase-1 is associated with poor prognosis in colorectal cancer. Nat Med 2(4):461–462CrossRefGoogle Scholar
  36. 36.
    Curran S, Murray GI (1999) Matrix metalloproteinases in tumour invasion and metastasis. J Pathol 189(3):300–308CrossRefGoogle Scholar
  37. 37.
    Lyall MS, Dundas SR, Curran S, Murray GI (2006) Profiling markers of prognosis in colorectal cancer. Clin Cancer Res 12(4):1184–1191CrossRefGoogle Scholar
  38. 38.
    Jensen SA, Vainer B, Bartels A, Brunner N, Sorensen JB (2010) Expression of matrix metalloproteinase 9 (MMP-9) and tissue inhibitor of metalloproteinases 1 (TIMP-1) by colorectal cancer cells and adjacent stroma cells--associations with histopathology and patients outcome. Eur J Cancer 46(18):3233–3242CrossRefGoogle Scholar
  39. 39.
    Chu D, Zhao Z, Zhou Y, Li Y, Li J, Zheng J, Zhao Q, Wang W (2012) Matrix metalloproteinase-9 is associated with relapse and prognosis of patients with colorectal cancer. Ann Surg Oncol 19(1):318–325CrossRefGoogle Scholar
  40. 40.
    Akishima-Fukasawa Y, Ishikawa Y, Akasaka Y, Uzuki M, Inomata N, Yokoo T, Ishii R, Shimokawa R, Mukai K, Kiguchi H, Suzuki K, Fujiwara M, Ogata K, Niino H, Sugiura H, Ichinose A, Kuroda Y, Kuroda D, Ishii T (2011) Histopathological predictors of regional lymph node metastasis at the invasive front in early colorectal cancer. Histopathology 59(3):470–481CrossRefGoogle Scholar
  41. 41.
    Gonzalez L, Eiro N, Gonzalez LO, Andicoechea A, Barbon E, Garcia-Muniz JL, Vizoso FJ (2012) Effect of the expression of matrix metalloproteases and their tissue inhibitors on survival of patients with resectable colorectal cancer. Dig Dis Sci 57(8):2063–2071CrossRefGoogle Scholar
  42. 42.
    Curran S, Dundas SR, Buxton J, Leeman MF, Ramsay R, Murray GI (2004) Matrix metalloproteinase/tissue inhibitors of matrix metalloproteinase phenotype identifies poor prognosis colorectal cancers. Clin Cancer Res 10(24):8229–8234CrossRefGoogle Scholar
  43. 43.
    Eiro N, Fernandez-Garcia B, Vazquez J, Del Casar JM, Gonzalez LO, Vizoso FJ (2015) A phenotype from tumor stroma based on the expression of metalloproteases and their inhibitors, associated with prognosis in breast cancer. Oncoimmunology 4(7):e992222CrossRefGoogle Scholar
  44. 44.
    Eiró N, Bermudez-Fernandez S, Fernandez-Garcia B, Atienza S, Beridze N, Escaf S, Vizoso FJ (2014) Analysis of the expression of interleukins, interferon β, and nuclear factor-κ B in prostate Cancer and their relationship with biochemical recurrence. J Immunother 37(7):366–373CrossRefGoogle Scholar
  45. 45.
    Fernandez-Garcia B, Eiró N, Marín L, González-Reyes S, González LO, Lamelas ML, Vizoso FJ (2014) Expression and prognostic significance of fibronectin and matrix metalloproteases in breast cancer metastasis. Histopathology 64(4):512–522CrossRefGoogle Scholar
  46. 46.
    González-Reyes S, Marín L, González L, González LO, del Casar JM, Lamelas ML, González-Quintana JM, Vizoso FJ (2010) Study of TLR3, TLR4 and TLR9 in breast carcinomas and their association with metastasis. BMC Cancer 10(1):665CrossRefGoogle Scholar
  47. 47.
    Nunez NG, Andreani V, Crespo MI, Nocera DA, Breser ML, Moron G, Dejager L, Libert C, Rivero V, Maccioni M (2012) IFNbeta produced by TLR4-activated tumor cells is involved in improving the antitumoral immune response. Cancer Res 72(3):592–603CrossRefGoogle Scholar
  48. 48.
    Huang B, Zhao J, Unkeless JC, Feng ZH, Xiong H (2008) TLR signaling by tumor and immune cells: a double-edged sword. Oncogene 27(2):218–224CrossRefGoogle Scholar
  49. 49.
    Merrell MA, Ilvesaro JM, Lehtonen N, Sorsa T, Gehrs B, Rosenthal E, Chen D, Shackley B, Harris KW, Selander KS (2006) Toll-like receptor 9 agonists promote cellular invasion by increasing matrix metalloproteinase activity. Mol Cancer Res 4(7):437–447CrossRefGoogle Scholar
  50. 50.
    Lee Y, Kim H, Kim S, Kim KH, Chung JH (2010) Activation of toll-like receptors 2, 3 or 5 induces matrix metalloproteinase-1 and -9 expression with the involvement of MAPKs and NF-kappaB in human epidermal keratinocytes. Exp Dermatol 19(8):e44–e49CrossRefGoogle Scholar
  51. 51.
    Agarwal S, Misra R, Aggarwal A (2010) Induction of metalloproteinases expression by TLR ligands in human fibroblast like synoviocytes from juvenile idiopathic arthritis patients. Indian J Med Res 131:771–779Google Scholar
  52. 52.
    Rath T, Stockle J, Roderfeld M, Tschuschner A, Graf J, Roeb E (2011) Matrix metalloproteinase-13 is regulated by toll-like receptor-9 in colorectal cancer cells and mediates cellular migration. Oncol Lett 2(3):483–488CrossRefGoogle Scholar
  53. 53.
    Eiro N, Fernandez-Gomez J, Sacristan R, Fernandez-Garcia B, Lobo B, Gonzalez-Suarez J, Quintas A, Escaf S, Vizoso FJ (2017) Stromal factors involved in human prostate cancer development, progression and castration resistance. J Cancer Res Clin Oncol 143(2):351–359CrossRefGoogle Scholar
  54. 54.
    Delebecq TJ, Porte H, Zerimech F, Copin MC, Gouyer V, Dacquembronne E, Balduyck M, Wurtz A, Huet G (2000) Overexpression level of stromelysin 3 is related to the lymph node involvement in non-small cell lung cancer. Clin Cancer Res 6(3):1086–1092Google Scholar
  55. 55.
    Zhao ZS, Chu YQ, Ye ZY, Wang YY, Tao HQ (2010) Overexpression of matrix metalloproteinase 11 in human gastric carcinoma and its clinicopathologic significance. Hum Pathol 41(5):686–696CrossRefGoogle Scholar
  56. 56.
    Kessenbrock K, Plaks V, Werb Z (2010) Matrix metalloproteinases: regulators of the tumor microenvironment. Cell 141(1):52–67CrossRefGoogle Scholar
  57. 57.
    Bhowmick NA, Neilson EG, Moses HL (2004) Stromal fibroblasts in cancer initiation and progression. Nature 432(7015):332–337CrossRefGoogle Scholar
  58. 58.
    Peruzzi D, Mori F, Conforti A, Lazzaro D, De Rinaldis E, Ciliberto G, La Monica N, Aurisicchio L (2009) MMP11: a novel target antigen for cancer immunotherapy. Clin Cancer Res 15(12):4104–4113CrossRefGoogle Scholar
  59. 59.
    McMillan DC, Hole DJ, McArdle CS (2008) The impact of old age on cancer-specific and non-cancer-related survival following elective potentially curative surgery for dukes a/B colorectal cancer. Br J Cancer 99(7):1046–1049CrossRefGoogle Scholar
  60. 60.
    Oliphant R, Horgan PG, Morrison DS, McMillan DC (2015) Validation of a modified clinical risk score to predict cancer-specific survival for stage II colon cancer. Cancer Med 4(1):84–89CrossRefGoogle Scholar
  61. 61.
    Lemmens VE, Janssen-Heijnen ML, Verheij CD, Houterman S, Repelaer van Driel OJ, Coebergh JW (2005) Co-morbidity leads to altered treatment and worse survival of elderly patients with colorectal cancer. Br J Surg 92(5):615–623CrossRefGoogle Scholar
  62. 62.
    Roxburgh CS, McMillan DC (2010) Role of systemic inflammatory response in predicting survival in patients with primary operable cancer. Future Oncol 6(1):149–163CrossRefGoogle Scholar
  63. 63.
    Turner N, Wong HL, Templeton A, Tripathy S, Whiti Rogers T, Croxford M, Jones I, Sinnathamby M, Desai J, Tie J, Bae S, Christie M, Gibbs P, Tran B (2016) Analysis of local chronic inflammatory cell infiltrate combined with systemic inflammation improves prognostication in stage II colon cancer independent of standard clinicopathologic criteria. Int J Cancer 138(3):671–678CrossRefGoogle Scholar

Copyright information

© Arányi Lajos Foundation 2019

Authors and Affiliations

  1. 1.Unidad de InvestigaciónFundación Hospital de JoveGijónSpain
  2. 2.Servicio de Cirugía GeneralFundación Hospital de JoveGijónSpain
  3. 3.Servicio de Anatomía PatológicaFundación Hospital de JoveGijónSpain

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