MMP-13 stimulates osteoclast differentiation and activation in tumour breast bone metastases.
BACKGROUND
The increased bone degradation in osteolytic metastases depends on stimulation of mature osteoclasts and on continuous differentiation of new pre-osteoclasts. Metalloproteinases (MMP)-13 is expressed in a broad range of primary malignant tumours and it is emerging as a novel biomarker. Recent data suggest a direct role of MMP-13 in dissolving bone matrix complementing the activity of MMP-9 and other enzymes. Tumour-microenvironment interactions alter gene expression in malignant breast tumour cells promoting osteolytic bone metastasis. Gene expression profiles revealed that MMP-13 was among the up-regulated genes in tumour-bone interface and its abrogation reduced bone erosion. The precise mechanism remained not fully understood. Our purpose was to further investigate the mechanistic role of MMP-13 in bone osteolytic lesions.


METHODS
MDA-MB-231 breast cancer cells that express MMP-13 were used as a model for in vitro and in vivo experiments. Conditioned media from MDA-MB-231 cells were added to peripheral blood mononuclear cultures to monitor pre-osteoclast differentiation and activation. Bone erosion was evaluated after injection of MMP-13-silenced MDA-MB-231 cells into nude mice femurs.


RESULTS
MMP-13 was co-expressed by human breast tumour bone metastases with its activator MT1-MMP. MMP-13 was up-regulated in breast cancer cells after in vitro stimulation with IL-8 and was responsible for increased bone resorption and osteoclastogenesis, both of which were reduced by MMP inhibitors. We hypothesized that MMP-13 might be directly involved in the loop promoting pre-osteoclast differentiation and activity. We obtained further evidence for a direct role of MMP-13 in bone metastasis by a silencing approach: conditioned media from MDA-MB-231 after MMP-13 abrogation or co-cultivation of silenced cells with pre-osteoclast were unable to increase pre-osteoclast differentiation and resorption activity. MMP-13 activated pre-MMP-9 and promoted the cleavage of galectin-3, a suppressor of osteoclastogenesis, thus contributing to pre-osteoclast differentiation. Accordingly, MMP-13 abrogation in tumour cells injected into the femurs of nude mice reduced the differentiation of TRAP positive cells in bone marrow and within the tumour mass as well as bone erosion.


CONCLUSIONS
These results indicate that within the inflammatory bone microenvironment MMP-13 production was up-regulated in breast tumour cells leading to increased pre-osteoclast differentiation and their subsequent activation.
Experimental Oncology 2, CRO, IRCCS, National Cancer Institute, Via Franco Gallini 2, 33081 Aviano (PN), Italy.
Eliana
E
Pivetta
Martina
M
Scapolan
Marina
M
Pecolo
Bruna
B
Wassermann
Imad
I
Abu-Rumeileh
Luca
L
Balestreri
Eugenio
E
Borsatti
Claudio
C
Tripodo
Alfonso
A
Colombatti
Paola
P
Spessotto
D000230:Adenocarcinoma / Q000378:metabolism / Q000473:pathology*; D000818:Animals; D001859:Bone Neoplasms / Q000378:metabolism / Q000473:pathology* / Q000556:secondary*; D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology*; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D060833:Cellular Microenvironment; D016207:Cytokines / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism; D005260:Female; D037502:Galectin 3 / Q000378:metabolism; D006801:Humans; D053509:Matrix Metalloproteinase 13 / Q000378:metabolism*; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D051379:Mice; D008819:Mice, Nude; D010010:Osteoclasts / Q000378:metabolism / Q000473:pathology*; D011498:Protein Precursors / Q000378:metabolism; D023041:Xenograft Model Antitumor Assays
D016207:Cytokines; D037502:Galectin 3; D011498:Protein Precursors; D053509:Matrix Metalloproteinase 13; D020780:Matrix Metalloproteinase 9