We investigated the effects of the matrix metalloproteinase 13 (MMP13)-selective inhibitor, 5-(4-{4-[4-(4-fluorophenyl)-1,3-oxazol-2？-yl]phenoxy}phenoxy)-5-(2-methoxyethyl)pyrimidine-2,4,6(1H,3H,5H)-trione (Cmpd-1), on the primary tumor growth and breast cancer-associated bone remodeling using xenograft and syngeneic mouse models. We used human breast cancer MDA-MB-231 cells inoculated into the mammary fat pad and left ventricle of BALB/c Nu/Nu mice, respectively, and spontaneously metastasizing 4T1.2-Luc mouse mammary cells inoculated into mammary fat pad of BALB/c mice. In a prevention setting, treatment with Cmpd-1 markedly delayed the growth of primary tumors in both models, and reduced the onset and severity of osteolytic lesions in the MDA-MB-231 intracardiac model. Intervention treatment with Cmpd-1 on established MDA-MB-231 primary tumors also significantly inhibited subsequent growth. In contrast, no effects of Cmpd-1 were observed on soft organ metastatic burden following intracardiac or mammary fat pad inoculations of MDA-MB-231 and 4T1.2-Luc cells respectively. MMP13 immunostaining of clinical primary breast tumors and experimental mice tumors revealed intra-tumoral and stromal expression in most tumors, and vasculature expression in all. MMP13 was also detected in osteoblasts in clinical samples of breast-to-bone metastases. The data suggest that MMP13-selective inhibitors, which lack musculoskeletal side effects, may have therapeutic potential both in primary breast cancer and cancer-induced bone osteolysis.
References
[1]
Martin MD, Matrisian LM (2007) The other side of MMPs: protective roles in tumor progression. Cancer Metastasis Rev 26: 717–724.
[2]
Wilson TJ, Singh RK (2008) Proteases as modulators of tumor-stromal interaction: primary tumors to bone metastases. Biochim Biophys Acta 1785: 85–95.
[3]
Overall CM, Kleifeld O (2006) Tumour microenvironment - opinion: validating matrix metalloproteinases as drug targets and anti-targets for cancer therapy. Nat Rev Cancer 6: 227–239.
[4]
Overall CM, Kleifeld O (2006) Towards third generation matrix metalloproteinase inhibitors for cancer therapy. Br J Cancer 94: 941–946.
[5]
Zhang B, Cao X, Liu Y, Cao W, Zhang F, et al. (2008) Tumor-derived matrix metalloproteinase-13 (MMP-13) correlates with poor prognoses of invasive breast cancer. BMC Cancer 8: 83.
[6]
Kominsky SL, Doucet M, Thorpe M, Weber KL (2008) MMP-13 is over-expressed in renal cell carcinoma bone metastasis and is induced by TGF-beta1. Clin Exp Metastasis 25: 865–870.
[7]
Hsu CP, Shen GH, Ko JL (2006) Matrix metalloproteinase-13 expression is associated with bone marrow microinvolvement and prognosis in non-small cell lung cancer. Lung Cancer 52: 349–357.
[8]
Leeman MF, McKay JA, Murray GI (2002) Matrix metalloproteinase 13 activity is associated with poor prognosis in colorectal cancer. J Clin Pathol 55: 758–762.
[9]
Tardif G, Reboul P, Pelletier JP, Martel-Pelletier J (2004) Ten years in the life of an enzyme: the story of the human MMP-13 (collagenase-3). Mod Rheumatol 14: 197–204.
[10]
Zigrino P, Kuhn I, Bauerle T, Zamek J, Fox JW, et al. (2009) Stromal expression of MMP-13 is required for melanoma invasion and metastasis. J Invest Dermatol 129: 2686–2693.
[11]
Lafleur MA, Drew AF, de Sousa EL, Blick T, Bills M, et al. (2005) Upregulation of matrix metalloproteinases (MMPs) in breast cancer xenografts: a major induction of stromal MMP-13. Int J Cancer 114: 544–554.
[12]
Inada M, Wang Y, Byrne MH, Rahman MU, Miyaura C, et al. (2004) Critical roles for collagenase-3 (Mmp13) in development of growth plate cartilage and in endochondral ossification. Proc Natl Acad Sci U S A 101: 17192–17197.
[13]
Stickens D, Behonick DJ, Ortega N, Heyer B, Hartenstein B, et al. (2004) Altered endochondral bone development in matrix metalloproteinase 13-deficient mice. Development 131: 5883–5895.
[14]
Cowan RW, Mak IW, Colterjohn N, Singh G, Ghert M (2009) Collagenase expression and activity in the stromal cells from giant cell tumour of bone. Bone 44: 865–871.
[15]
Morgia G, Falsaperla M, Malaponte G, Madonia M, Indelicato M, et al. (2005) Matrix metalloproteinases as diagnostic (MMP-13) and prognostic (MMP-2, MMP-9) markers of prostate cancer. Urol Res 33: 44–50.
[16]
Ohshiba T, Miyaura C, Inada M, Ito A (2003) Role of RANKL-induced osteoclast formation and MMP-dependent matrix degradation in bone destruction by breast cancer metastasis. Br J Cancer 88: 1318–1326.
[17]
Nannuru KC, Futakuchi M, Varney ML, Vincent TM, Marcusson EG, et al. (2010) Matrix metalloproteinase (MMP)-13 regulates mammary tumor-induced osteolysis by activating MMP9 and transforming growth factor-beta signaling at the tumor-bone interface. Cancer Res 70: 3494–3504.
[18]
Morrison C, Mancini S, Cipollone J, Kappelhoff R, Roskelley C, et al. (2011) Microarray and proteomic analysis of breast cancer cell and osteoblast co-cultures: role of osteoblast matrix metalloproteinase (MMP)-13 in bone metastasis. J Biol Chem 286: 34271–34285.
[19]
Tuckermann JP, Pittois K, Partridge NC, Merregaert J, Angel P (2000) Collagenase-3 (MMP-13) and integral membrane protein 2a (Itm2a) are marker genes of chondrogenic/osteoblastic cells in bone formation: sequential temporal, and spatial expression of Itm2a, alkaline phosphatase, MMP-13, and osteocalcin in the mouse. J Bone Miner Res 15: 1257–1265.
[20]
Hattori N, Mochizuki S, Kishi K, Nakajima T, Takaishi H, et al. (2009) MMP-13 plays a role in keratinocyte migration, angiogenesis, and contraction in mouse skin wound healing. Am J Pathol 175: 533–546.
[21]
Uchinami H, Seki E, Brenner DA, D'Armiento J (2006) Loss of MMP 13 attenuates murine hepatic injury and fibrosis during cholestasis. Hepatology 44: 420–429.
[22]
Neuhold LA, Killar L, Zhao W, Sung ML, Warner L, et al. (2001) Postnatal expression in hyaline cartilage of constitutively active human collagenase-3 (MMP-13) induces osteoarthritis in mice. J Clin Invest 107: 35–44.
[23]
Wernicke D, Seyfert C, Gromnica-Ihle E, Stiehl P (2006) The expression of collagenase 3 (MMP-13) mRNA in the synovial tissue is associated with histopathologic type II synovitis in rheumatoid arthritis. Autoimmunity 39: 307–313.
[24]
Little CB, Barai A, Burkhardt D, Smith SM, Fosang AJ, et al. (2009) Matrix metalloproteinase 13-deficient mice are resistant to osteoarthritic cartilage erosion but not chondrocyte hypertrophy or osteophyte development. Arthritis Rheum 60: 3723–3733.
[25]
Lovejoy B, Welch AR, Carr S, Luong C, Broka C, et al. (1999) Crystal structures of MMP-1 and -13 reveal the structural basis for selectivity of collagenase inhibitors. Nat Struct Biol 6: 217–221.
[26]
Blagg JA, Noe MC, Wolf-Gouveia LA, Reiter LA, Laird ER, et al. (2005) Potent pyrimidinetrione-based inhibitors of MMP-13 with enhanced selectivity over MMP-14. Bioorg Med Chem Lett 15: 1807–1810.
[27]
Reiter LA, Freeman-Cook KD, Jones CS, Martinelli GJ, Antipas AS, et al. (2006) Potent, selective pyrimidinetrione-based inhibitors of MMP-13. Bioorg Med Chem Lett 16: 5822–5826.
[28]
Eckhardt BL, Parker BS, van Laar RK, Restall CM, Natoli AL, et al. (2005) Genomic analysis of a spontaneous model of breast cancer metastasis to bone reveals a role for the extracellular matrix. Mol Cancer Res 3: 1–13.
[29]
Lelekakis M, Moseley JM, Martin TJ, Hards D, Williams E, et al. (1999) A novel orthotopic model of breast cancer metastasis to bone. Clin Exp Metastasis 17: 163–170.
[30]
Tester AM, Ruangpanit N, Anderson RL, Thompson EW (2000) MMP-9 secretion and MMP-2 activation distinguish invasive and metastatic sublines of a mouse mammary carcinoma system showing epithelial-mesenchymal transition traits. Clin Exp Metastasis 18: 553–560.
[31]
Sharp JA, Waltham M, Williams ED, Henderson MA, Thompson EW (2004) Transfection of MDA-MB-231 human breast carcinoma cells with bone sialoprotein (BSP) stimulates migration and invasion in vitro and growth of primary and secondary tumors in nude mice. Clin Exp Metastasis 21: 19–29.
[32]
Tester AM, Sharp JA, Dhanesuan N, Waltham M, Thompson EW (2002) Correlation between extent of osteolytic damage and metastatic burden of human breast cancer metastasis in nude mice: real-time PCR quantitation. Clin Exp Metastasis 19: 377–383.
[33]
Ory B, Heymann MF, Kamijo A, Gouin F, Heymann D, et al. (2005) Zoledronic acid suppresses lung metastases and prolongs overall survival of osteosarcoma-bearing mice. Cancer 104: 2522–2529.
[34]
Udabage L, Brownlee GR, Waltham M, Blick T, Walker EC, et al. (2005) Antisense-mediated suppression of hyaluronan synthase 2 inhibits the tumorigenesis and progression of breast cancer. Cancer Res 65: 6139–6150.
[35]
Filgueira L (2004) Fluorescence-based staining for tartrate-resistant acidic phosphatase (TRAP) in osteoclasts combined with other fluorescent dyes and protocols. J Histochem Cytochem 52: 411–414.
[36]
Thiolloy S, Halpern J, Holt GE, Schwartz HS, Mundy GR, et al. (2009) Osteoclast-derived matrix metalloproteinase-7, but not matrix metalloproteinase-9, contributes to tumor-induced osteolysis. Cancer Res 69: 6747–6755.
[37]
Nuti E, Tuccinardi T, Rossello A (2007) Matrix metalloproteinase inhibitors: new challenges in the era of post broad-spectrum inhibitors. Curr Pharm Des 13: 2087–2100.
[38]
Peterson JT (2006) The importance of estimating the therapeutic index in the development of matrix metalloproteinase inhibitors. Cardiovasc Res 69: 677–687.
[39]
Johnson AR, Pavlovsky AG, Ortwine DF, Prior F, Man CF, et al. (2007) Discovery and characterization of a novel inhibitor of matrix metalloprotease-13 that reduces cartilage damage in vivo without joint fibroplasia side effects. J Biol Chem 282: 27781–27791.
[40]
Knauper V, Lopez-Otin C, Smith B, Knight G, Murphy G (1996) Biochemical characterization of human collagenase-3. J Biol Chem 271: 1544–1550.
[41]
Bergers G, Javaherian K, Lo KM, Folkman J, Hanahan D (1999) Effects of angiogenesis inhibitors on multistage carcinogenesis in mice. Science 284: 808–812.
[42]
Nielsen BS, Rank F, Lopez JM, Balbin M, Vizoso F, et al. (2001) Collagenase-3 expression in breast myofibroblasts as a molecular marker of transition of ductal carcinoma in situ lesions to invasive ductal carcinomas. Cancer Res 61: 7091–7100.
[43]
Lederle W, Hartenstein B, Meides A, Kunzelmann H, Werb Z, et al. (2010) MMP13 as a stromal mediator in controlling persistent angiogenesis in skin carcinoma. Carcinogenesis 31: 1175–1184.
[44]
Behonick DJ, Xing Z, Lieu S, Buckley JM, Lotz JC, et al. (2007) Role of matrix metalloproteinase 13 in both endochondral and intramembranous ossification during skeletal regeneration. PLoS ONE 2: e1150.
[45]
Lynch CC (2011) Matrix metalloproteinases as master regulators of the vicious cycle of bone metastasis. Bone 48: 44–53.
[46]
Andersen TL, del Carmen Ovejero M, Kirkegaard T, Lenhard T, Foged NT, et al. (2004) A scrutiny of matrix metalloproteinases in osteoclasts: evidence for heterogeneity and for the presence of MMPs synthesized by other cells. Bone 35: 1107–1119.
[47]
Krane SM, Inada M (2008) Matrix metalloproteinases and bone. Bone 43: 7–18.
[48]
Scott DK, Brakenhoff KD, Clohisy JC, Quinn CO, Partridge NC (1992) Parathyroid hormone induces transcription of collagenase in rat osteoblastic cells by a mechanism using cyclic adenosine 3′,5′-monophosphate and requiring protein synthesis. Mol Endocrinol 6: 2153–2159.
[49]
Shah R, Alvarez M, Jones DR, Torrungruang K, Watt AJ, et al. (2004) Nmp4/CIZ regulation of matrix metalloproteinase 13 (MMP-13) response to parathyroid hormone in osteoblasts. Am J Physiol Endocrinol Metab 287: E289–296.
[50]
Delaisse JM, Andersen TL, Engsig MT, Henriksen K, Troen T, et al. (2003) Matrix metalloproteinases (MMP) and cathepsin K contribute differently to osteoclastic activities. Microsc Res Tech 61: 504–513.
[51]
Hill PA, Murphy G, Docherty AJ, Hembry RM, Millican TA, et al. (1994) The effects of selective inhibitors of matrix metalloproteinases (MMPs) on bone resorption and the identification of MMPs and TIMP-1 in isolated osteoclasts. J Cell Sci 107(Pt 11): 3055–3064.
[52]
Sato T, del Carmen Ovejero M, Hou P, Heegaard AM, Kumegawa M, et al. (1997) Identification of the membrane-type matrix metalloproteinase MT1-MMP in osteoclasts. J Cell Sci 110(Pt 5): 589–596.
[53]
Tezuka K, Tezuka Y, Maejima A, Sato T, Nemoto K, et al. (1994) Molecular cloning of a possible cysteine proteinase predominantly expressed in osteoclasts. J Biol Chem 269: 1106–1109.
[54]
Winter MC, Holen I, Coleman RE (2008) Exploring the anti-tumour activity of bisphosphonates in early breast cancer. Cancer Treat Rev 34: 453–475.
[55]
Sasaki A, Boyce BF, Story B, Wright KR, Chapman M, et al. (1995) Bisphosphonate risedronate reduces metastatic human breast cancer burden in bone in nude mice. Cancer Res 55: 3551–3557.
[56]
Migliorati CA, Siegel MA, Elting LS (2006) Bisphosphonate-associated osteonecrosis: a long-term complication of bisphosphonate treatment. Lancet Oncol 7: 508–514.
