Bone metastases occur in a high proportion of breast cancer patients with metastatic disease and are associated with severe morbidity and eventual mortality when visceral organs become involved. Dissecting the molecular mechanisms involved in tumor cell survival and outgrowth in bone is essential for the development of targeted therapies to reduce patient mortality. Here we report that tumor-derived cathepsin B is a key contributor to bone metastasis. Our data demonstrate for the first time that selective inhibition of cathepsin B using small molecule inhibitors significantly reduces metastasis and has therapeutic potential.
There are a number of critical steps required for growth of tumors in bone. This includes collaboration between the tumor cells themselves and the bone stroma for stimulating angiogenesis, invasion through the bone matrix and growth beyond the micrometastatic stage. Cathepsin B upregulation has been documented in several human cancers, including breast, prostate and melanoma (15
). Our data demonstrate that cathepsin B has critical tumor-specific functions in breast cancer metastasis that are completely independent from primary tumor growth. We show that tumor cells themselves can directly degrade the major bone matrix protein collagen I and that cathepsin B is important, if not essential, in this process. Previous studies have implicated tumor cell cathepsin B in degradation of ECM proteins including fibronectin, laminin and collagen IV (36
). Additionally, and in support of our studies, inhibition of cathepsin B has been demonstrated to reduce collagen I degradation by prostate carcinoma cell lines (38
), again suggestive of a tumor cell-specific role in bone lysis. Consistent with a role in invasion, we demonstrated a critical function of tumor-derived cathepsin B in metastasis in the 4T1.2 model. Although orthotopic tumor growth was not altered by cathepsin B knockdown, pulmonary and bone (spine) metastases were significantly reduced. As outgrowth of disseminated tumor cells into macrometastases requires multiple steps including invasion through secondary tissues, preventing ECM degradation by cathepsin B inhibition is likely to reduce metastatic burden.
Cathepsin B has also been implicated in stromal cell-associated pro-tumorigenic functions. In breast cancer, this has been demonstrated using the MMTV-PyMT model where host-derived cathepsin B promotes lung metastasis (39
). The expression of cathepsin B predominantly in tumor-infiltrating macrophages indicated that macrophage derived cathepsin B contributed to metastasis. The expression of cathepsin B in tumor-associated endothelial cells and macrophages has been associated with tumor progression via promotion of angiogenesis (17
). Cathepsin B has also been reported to be expressed in stromal fibroblasts and macrophages in colon and prostate cancers (40
). Taken together, cathepsin B has key roles in tumor progression, by invasion through the ECM and stimulation of angiogenesis. Our treatment of 4T1.2-tumor bearing mice with the selective small molecule cathepsin B inhibitor CA-074 enabled inhibition of both tumor and stromal cathepsin B and allowed assessment of the therapeutic benefit of inhibiting this protease in vivo
. Consistent with cathepsin B transcript knockdown, treatment with CA-074 did not alter primary tumor growth yet suppression of metastasis was impressive. In fact, bone metastases could not be detected histologically in tumor-bearing mice treated with the inhibitor, an impressive result considering the aggressive nature of 4T1.2 tumors. Due to the role of cathepsin B in degradation of collagen IV and I, we hypothesized that metastasis inhibition was caused by decreased invasion at both the primary and metastatic site. Our results in a late treatment setting support a role for cathepsin B in metastasis post tumor cell arrest in distant tissues. Commencement of CA-074 treatment post primary tumor resection significantly decreased metastatic growth in lung and bone. The use of cathepsin inhibitors to decrease spontaneous breast cancer metastasis to bone from the mammary gland has not been reported previously. However, use of cathepsin K (42
) (another cysteine cathepsin) and cathepsin G (43
) (a serine protease) inhibitors have been documented to suppress the formation and growth of breast cancer osteolytic lesions in experimental models of metastasis.
The therapeutic benefit of CA-074 treatment in the 4T1.2 model was in contrast to the broad spectrum inhibitor JPM-OEt, which did not decrease lung or spine metastases. In agreement with our studies, treatment of polyoma middle T oncogene-induced mammary carcinomas with JPM-OEt did not alter tumor weights and lung metastasis (33
). Together, these studies reveal the importance of using specific targeted therapies in vivo.
JPM-OEt targets several cysteine cathepsins, some of which could actually have tumor suppressive functions. For example, cathepsin L activity is decreased in highly metastatic 4T1.2 tumors compared to weakly or non-metastatic tumors (20
) suggesting inhibitory effects of this protease on tumor progression, as has been suggested before in skin tumorigenesis (44
). The importance of using specific protease inhibitors is clear from early clinical trials using MMP inhibitors where inhibition of certain MMPs actually has deleterious effects in patients (45
). These studies highlight the significant effort needed to determine which specific proteases contribute to advanced disease progression before potential drugs can be tested in the clinic.
Current treatments available for patients with bone metastasis are aimed primarily at reducing the morbidity associated with bone lysis, a hallmark of breast cancer bone disease. One target of such treatments is cathepsin K. Cathepsin K is expressed predominantly in osteoclasts where it is secreted and has a key role in bone proteolysis, including degradation of collagen I, osteonectin and osteopontin (47
). The potential role of cathepsin K in osteolytic bone tumors is evident from its expression in tumor associated osteoclasts (48
), in breast and prostate metastases in bone and in giant cell tumor of bone (49
). Cathepsin K inhibitors block bone resorption (50
), reducing the pain associated with osteolytic disease, and in turn slow the growth of bone tumors. However, as with the use of bisphosphonates that also suppress osteolysis, treatment is not curative. Therefore, an ideal treatment (or combination of treatments) is one that targets both the tumor cell and the associated stromal populations that promote osteolysis, angiogenesis and tumor growth. The fact that cathepsin B has essential roles throughout the metastatic cascade, in tumor and stromal cells, suggests that combining cathepsin B inhibitors with conventional therapies may be of clinical value.