title: Elevated ratio of MMP2/MMP9 activity is associated with poor response to chemotherapy in osteosarcoma.
abstract: BACKGROUND
  Matrix metalloproteinases (MMPs) are crucially involved in the regulation of multiple stages of cancer progression. Elevated MMP levels have been associated with the development of metastases and poor prognosis in several types of cancer. However, the role of MMPs in osteosarcoma and their prognostic value is still unclear. Available data are conflicting, most likely due to different technical approaches. We hypothesized that in contrast to total mRNA or protein levels frequently analyzed in previous studies the enzymatic activities of MMPs and their inhibitors the tissue inhibitors of matrix metalloproteinases (TIMPs) are closer related to their biological functions. We therefore aimed to evaluate the reliability of different zymography techniques for the quantification of MMP and TIMP activities in osteosarcoma biopsies in order to investigate their distribution, possible regulation and prognostic value.
  
  
  METHODS
  All analyses were done using cryo-conserved osteosarcoma pretreatment biopsies (n = 18). Gene and protein expression of MMPs and TIMPs were analyzed by RT-qPCR and western blot analysis, respectively. Overall MMP activity was analyzed by in situ zymography, individual MMP activities were analyzed by gelatin zymography. Reverse zymography was used to detect and quantify TIMP activities.
  
  
  RESULTS
  Strong overall MMP activities could be detected in osteosarcoma pretreatment biopsies with MMP2 and MMP9 as predominant active MMPs. In contrast to total RNA or protein expression MMP2 and MMP9 activities showed significant quantitative differences between good and poor responders. While MMP9 activity was high in the good responder group and significantly decreased in the poor responder group, MMP2 activity showed a reverse distribution. Likewise, significant differences were detected concerning the activity of TIMPs resulting in a negative correlation of TIMP1 activity with MMP2 activity (p = 0.044) and negative correlations of TIMP2 and TIMP3 with MMP9 activity (p = 0.007 and p = 0.006).
  
  
  CONCLUSIONS
  In contrast to mRNA or protein levels MMP and TIMP activities showed significant differences between the analyzed good and poor responder groups. A shift from MMP9 to predominant MMP2 activity is associated with poor response to chemotherapy suggesting that the ratio of MMP2/MMP9 activity might be a valuable and easily accessible marker to predict the response to chemotherapy in osteosarcoma.
authors.affiliation: Clinic for Orthopedics and Trauma Surgery/Spinal Cord Injury Center; Center for Orthopedics, Trauma Surgery and Spinal Cord Injury, University Hospital Heidelberg, Heidelberg, Germany.
authors.forename: Pierre
authors.initials: P
authors.lastname: Kunz
authors.affiliation: Clinic for Orthopedics and Trauma Surgery/Spinal Cord Injury Center; Center for Orthopedics, Trauma Surgery and Spinal Cord Injury, University Hospital Heidelberg, Heidelberg, Germany.
authors.forename: Heiner
authors.initials: H
authors.lastname: Sähr
authors.affiliation: Clinic for Orthopedics and Trauma Surgery/Spinal Cord Injury Center; Center for Orthopedics, Trauma Surgery and Spinal Cord Injury, University Hospital Heidelberg, Heidelberg, Germany.
authors.forename: Burkhard
authors.initials: B
authors.lastname: Lehner
authors.affiliation: Clinic for Orthopedics and Trauma Surgery/Spinal Cord Injury Center; Center for Orthopedics, Trauma Surgery and Spinal Cord Injury, University Hospital Heidelberg, Heidelberg, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Fischer
authors.affiliation: Clinic for Orthopedics and Trauma Surgery/Spinal Cord Injury Center; Center for Orthopedics, Trauma Surgery and Spinal Cord Injury, University Hospital Heidelberg, Heidelberg, Germany.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Seebach
authors.affiliation: Clinic for Orthopedics and Trauma Surgery/Spinal Cord Injury Center; Center for Orthopedics, Trauma Surgery and Spinal Cord Injury, University Hospital Heidelberg, Heidelberg, Germany. Joerg.Fellenberg@med.uni-heidelberg.de.
authors.forename: Jörg
authors.initials: J
authors.lastname: Fellenberg
mesh_terms: D001706:Biopsy; D002945:Cisplatin / Q000008:administration & dosage; D004317:Doxorubicin / Q000008:administration & dosage; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D008297:Male; D020778:Matrix Metalloproteinase 2 / Q000096:biosynthesis* / Q000235:genetics; D020780:Matrix Metalloproteinase 9 / Q000096:biosynthesis* / Q000235:genetics; D008727:Methotrexate / Q000008:administration & dosage; D012516:Osteosarcoma / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology; D011379:Prognosis; D012333:RNA, Messenger / Q000235:genetics; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000096:biosynthesis* / Q000235:genetics; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000096:biosynthesis* / Q000235:genetics; D019717:Tissue Inhibitor of Metalloproteinase-3 / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D012333:RNA, Messenger; C560822:TIMP1 protein, human; C560823:TIMP2 protein, human; C088390:TIMP3 protein, human; D019715:Tissue Inhibitor of Metalloproteinase-1; D019717:Tissue Inhibitor of Metalloproteinase-3; D019716:Tissue Inhibitor of Metalloproteinase-2; D004317:Doxorubicin; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9; D002945:Cisplatin; D008727:Methotrexate
keywords: Chemotherapy; Matrix metalloproteinases; Osteosarcoma; Prognosis; Tissue inhibitor of matrix metalloproteinases; Zymography