In this report we provide evidence that elevated expression of MMP-1 contributes to the brain colonizing potential of human breast cancer cells in xenograft models of cancer progression.
Members of the MMP family play important roles in normal and malignant processes. Their functions in invasion and metastasis have been associated primarily with degradation of ECM components [18
]. In recent years, however, it has become increasingly clear that MMP substrates extend to many non-matrix extracellular and membrane-bound proteins, including protease precursors and inhibitors, cytokines, latent growth factors, growth factor-binding proteins, and adhesion molecules [17
]. Understanding how MMP-1 and other members of the MMP family promote metastasis, in part by altering the signaling milieu in the tissue microenvironment colonized by disseminating cells may be crucial for developing more effective therapies for metastatic cancer.
Our study shows that MMP-1 is highly expressed by the brain metastasis-derived variants of the human MDA-MB-231 breast cancer cell line; this supports findings reported by others [4
]. What drives the elevated expression remains to be established; the 231-BR and 231-BR3 variants have constitutive activation of STAT3, which has been linked to elevated expression of various genes, including VEGF, cyclin D and survivin [27
]. Transcriptional regulators of MMP-1 in cancer cells include STAT3 [28
] and members of the AP-1 family of transcription factors [29
Reducing the expression of MMP-1 with shRNA attenuated tumor growth in the mammary fat pads and reduced invasion through matrix-coated filters of the 231-BR and 231-BR3 cells, similar to the findings of other investigators using non-selected MDA-MB-231 cells [30
]. Metastatic lesions formed by cells expressing shRNA to MMP-1 in the lungs or brains, from i.v. or intra-cardiac injections, respectively, were smaller and fewer than those formed by the control cells. Without using a method to follow the fate of cells after injection into mice we cannot discern whether the reduction in metastasis number is due to impaired arrest and extravasation, or reduced proliferation in the metastatic site; the data would support a combination of these possibilities. MRI has been used to document the fate of MDA-MB-231-BR cells tagged with iron oxide particles in the brains of nude mice after intra-cardiac injection. The majority of the cells were rapidly eliminated, and only a small fraction of the initial inoculum formed actively growing metastases [32
]. Fitzgerald et al.
] reported high proliferation rates of brain metastases of this cell line, as we also found for metastases of control shRNA-expressing cells, while significantly fewer cells in brain metastases of the MMP-1 silenced cell lines were proliferating (Ki67-positive).
Our data show that MMP-1 can regulate the levels of TGF-α in culture supernatants of the MDA-MB-231-BR and -BR3 cells, which in turns affects activity of EGFR in the cancer cells. The activation of EGFR can regulate a wide variety of cellular functions [34
]. One related to brain metastasis is the recent report of EGF promoting heparanase function and Topoisomerase I localization in brain metastasizing breast cancer cells [36
]. Treatment with cetuximab, a humanized antibody to EGFR, reduced transmigration through a simulated blood–brain barrier and extended survival of mice injected with brain-colonizing breast cancer cells [4
]. MDA-MB-231-BR cells express phosphorylated EGFR in vivo
, and treatment with lapatinib, a small molecule tyrosine kinase inhibitor of EGFR and HER2, significantly reduced the numbers of large brain metastases formed by these cells [37
]. The TGF-α released around MMP-1 expressing cells may also have paracrine functions in the brain microenvironment, including induction of angiogenesis and neurogenesis [38
], and activation of astrocytes in response to injury [39
]. Reactive microglial and astrocytic responses to brain metastases have been reported in studies using the MDA-MB-231-BR model [33
] and other experimental brain metastasis models [41
], resembling the peritumoral changes seen in clinical brain metastases [43
]. These responses may promote the proliferation and survival of the metastatic cells [33
]. Reactive astrocytes have neuroprotective functions, which may be exploited by cancer cells; co-culture of astrocytes with brain metastatic cells protected the latter from chemotherapy-induced apoptosis, an effect dependent upon gap-junction communications between the different cell types [5
While not explored further in this study, MMP-1 activation of protease-activated receptor 1 (PAR1) may also contribute to the process of brain metastasis. Protease-activated receptors are members of the G protein coupled receptor family that are activated upon cleavage of an N-terminal tethered ligand. Thrombin and MMP-1 both activate PAR1, but MMP-1 is reported to cleave the tethered ligand at a unique site [46
]. PAR1 expression on breast cancer cells has been associated with a high metastatic potential, and inhibiting the downstream signals from PAR1, using a small molecule inhibitor, suppressed Akt-mediated survival pathways, and attenuated tumor growth and experimental lung metastasis [47
]. The brain metastatic variants of MDA-MB-231 maintain the high expression of PAR1 reported by others for the original cell line (Liu and Price, unpublished). PAR1 is also expressed by other cell types present in the brain microenvironment, including endothelial cells [48
] and astrocytes; activation of PAR1 on the latter can trigger astrogliosis [49