Novel MMP-9 substrates in cancer cells revealed by a label-free quantitative proteomics approach.
Matrix metalloproteinase-9 (MMP-9) is implicated in tumor metastasis as well as a variety of inflammatory and pathological processes. Although many substrates for MMP-9, including components of the extracellular matrix, soluble mediators such as chemokines, and cell surface molecules have been identified, we undertook a more comprehensive proteomics-based approach to identify new substrates to further understand how MMP-9 might contribute to tumor metastasis. Previous proteomics approaches to identify protease substrates have depended upon differential labeling of each sample. Instead we used a label-free quantitative proteomics approach based on ultraperformance LC-ESI-high/low collision energy MS. Conditioned medium from a human metastatic prostate cancer cell line, PC-3ML, in which MMP-9 had been down-regulated by RNA interference was compared with that from the parental cells. From more than 200 proteins identified, 69 showed significant alteration in levels after depletion of the protease (>+/-2-fold), suggesting that they might be candidate substrates. Levels of six of these (amyloid-beta precursor protein, collagen VI, leukemia inhibitory factor, neuropilin-1, prostate cancer cell-derived growth factor (PCDGF), and protease nexin-1 (PN-1)) were tested in the conditioned media by immunoblotting. There was a strong correlation between results by ultraperformance LC-ESI-high/low collision energy MS and by immunoblotting giving credence to the label-free approach. Further information about MMP-9 cleavage was obtained by comparison of the peptide coverage of collagen VI in the presence and absence of MMP-9 showing increased sensitivity of the C- and N-terminal globular regions over the helical regions. Susceptibility of PN-1 and leukemia inhibitory factor to MMP-9 degradation was confirmed by in vitro incubation of the recombinant proteins with recombinant MMP-9. The MMP-9 cleavage sites in PN-1 were sequenced. This study provides a new label-free method for degradomics cell-based screening leading to the identification of a series of proteins whose levels are affected by MMP-9, some of which are clearly direct substrates for MMP-9 and become candidates for involvement in metastasis.
Xu
Danmei
D
Radiation Oncology and Biology, Medical Science Division, Churchill Hospital, University of Oxford, Oxford OX3 7LJ, United Kingdom.
Suenaga
Naoko
N
Edelmann
Mariola J
MJ
Fridman
Rafael
R
Muschel
Ruth J
RJ
Kessler
Benedikt M
BM
D000595:Amino Acid Sequence; D016564:Amyloid beta-Protein Precursor / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D045744:Cell Line, Tumor; D017077:Culture Media, Conditioned; D006801:Humans; D053605:Leukemia Inhibitory Factor / Q000378:metabolism; D008297:Male; D020780:Matrix Metalloproteinase 9 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D061965:Matrix Metalloproteinase Inhibitors; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011471:Prostatic Neoplasms / Q000201:enzymology* / Q000235:genetics; D058255:Protease Nexins; D040901:Proteomics / Q000379:methods; D034622:RNA Interference; D011956:Receptors, Cell Surface / Q000378:metabolism; D058258:Serpin E2; D013379:Substrate Specificity
C545487:APP protein, human; D016564:Amyloid beta-Protein Precursor; D017077:Culture Media, Conditioned; C507837:LIF protein, human; D053605:Leukemia Inhibitory Factor; D061965:Matrix Metalloproteinase Inhibitors; D058255:Protease Nexins; D011956:Receptors, Cell Surface; C545486:SERPINE2 protein, human; D058258:Serpin E2; D020780:Matrix Metalloproteinase 9