The Role of Cathepsins in Primary Bone Cancer and Metastases to the Bone
The bone microenvironment is hospitable to bone tumor development and growth, as well as being a favored site for the metastases of cancers such as prostate and breast. Cathepsins play a key role in the malignant progression at this site because their secretion within the bone matrix enables the expansion of neoplastic cells and leads to the release of active growth factors that promote cancer cell growth [
24,
135]. Referred to as the ’vicious cycle’, the secretion of proteolytic enzymes from neoplastic cells, and the resulting hyperactive remodeling and release of pro-tumorigenic factors from the bone matrix, is critical to tumor progression in the bone [
20]. The release of these growth factors constitutes one of the key mechanisms in the bone’s remodeling, allowing the physiological coupling between osteoblasts and the osteoclasts [
136]. Neoplastic cells also secrete bone-remodeling cytokines and growth factors such as PTH related peptide (PTHrP), and IL -1, -6,-8, and -11 into the bone microenvironment, which, in turn, act on osteoblastic stromal cells to enhance the production of osteoclast-activating factors such as RANKL [
20,
78,
137,
138,
139]. Additional tumor-derived growth factors, such as transforming growth factor- β (TGF-β), IGF-I, and IGF-II, as well as collagen type I peptides, also serve to attract tumor cells to the bone matrix, induce mitosis in tumor cells, and promote bone formation and remodeling by enhancing osteoclastogenesis [
14,
15,
24,
25,
137,
140,
141,
142,
143]. The endogenous cathepsin inhibitor, cystatin C, inhibits TGF-beta signaling in normal and neoplastic cells [
144]. Stromal and tumor microenvironmental conditions, such as acidosis within the bone, has been shown to increase osteoclasts’ function and resorption pit formation, which results in the release of multiple proteases: specifically members of the cysteine cathepsin family, such as cathepsins B, D, K and L [
58,
145,
146].
Several studies have shown the importance of cathepsins for the invasion and metastasis of OS cell lines [
43,
46,
48]. In normal human osteoblast cells and the OS cell line MG-63, the active secretion and RNA/protein production of CatB is generated in the presence of bone-resorbing agents such as IL-1β and PTH [
43]. In malignant bone tumors, CatB demonstrates activity at the tumor–bone interface with low levels of CatB detected around the bone [
110]. CatB is most extensively found at the periphery of primary tumors, correlating to its role in tumor invasion and the degradation of the ECM [
147]. The ability of CatB to degrade other matrix proteins and activate additional proteases is associated with neoplastic cell invasion and metastasis. Contrary to these findings, Husmann et al., discovered that—in comparison to the non-metastatic parental line Saos-2—the expression of the mature form of CatB in LM5 and LM7 cells was decreased by approximately 50% [
55].
Intriguingly, CatB has been discovered to also demonstrate anticancer effects in the OS cell line MG-63, which is mediated through the activation of the DR-5/p53/Bax/caspase-9/-3 signaling pathway and the DR-5/FADD/caspase-8/lysosomal/cathepsin B/caspase-3 signaling pathway [
148]. The release of CatB from the lysosome into the cytosol induces cell death in vitro [
44]. Caspase-8 mediated Bid cleavage leads to the permeabilization of the mitochondrial outer membrane and the release of cytochrome C [
149]. In the human OS cell line U2OS, CatB activity is activated after exposure to tumor necrosis factor-related apoptosis inducing ligand (TRAIL). CatB contributes to TRAIL-mediated apoptosis in human cells through Bid cleavage [
149,
150,
151]. CatB activity is significantly increased following 1–2 h of exposure to TRAIL [
149]. Triptolide inhibits cell viability and proliferation, and induced apoptosis in OS cell line MG-63 [
148]. Previous studies by Owa et al., support this finding, citing that triptolide induces lysosomal-mediated programmed cell death in MCF-7 BCa cells through CatB and caspase-3 [
152]. It is believed that the upregulation of CatB increases caspase 3 cleavage and induced apoptosis. During the early stages of apoptosis, cytosolic levels of CatB are increased [
148]. Triptolide increases lysosomal membrane permeability, causing a leakage of CatB into the cytosol within the first 3 h of treatment, triggering the apoptotic cascade. Triptolide increases DR-5, Bax, p53, and CatB proteins, as well as caspase-3,-8, and-9 activity [
148]. Clinically, elevated levels of CatB and CatL have been shown to be significant predictors of relapse and death for BCa patients [
116,
117,
153,
154]. Approximately 77% of BCa samples express CatB [
116]. The expression of CatB was also associated with the expression of CatD and CatL in BCa patient samples [
116]. Interestingly, high levels of CatB protein and activity were found in DU145, PC3, and LNCaP bone tumors in severe combined immunodeficiency (SCID) mice, despite the low CatB expression in PC3 and LNCaP cell lines in vitro [
155]. The use of activity-based probes provides a non-invasive modality in order to monitor cathepsin activity and the therapeutic efficacy of cathepsin-targeted agents. In metastatic bone cancer, CatB demonstrated a 2-fold increase compared to normal fibroblasts [
156]. CatB is higher in MDSCs isolated from the bone marrow of 4T1.2 tumor-bearing mice compared to those taken from lungs, which further suggests a differential regulation of MDSC-derived CatB within the bone microenvironment [
106]. BMV109, a pan-cysteine cathepsin probe, monitors for CatB and CatL [
157]. BMV109+ cells were expressed to similar extents in primary tumors derived from BCa cells 67NR (non-metastatic) and 4T1.2 (metastatic) [
95]. However, in mice which developed bone metastasis, 4T1.2 tumors exhibited a strong increase in cathepsin expression/activity compared to mice bearing 67NR tumors, suggesting that cathepsin activity is upregulated during metastasis [
95]. Withana et al. also demonstrated a decrease in CatB activity after treatment with CA-074 using the GB123 activity-based probe [
157].
In patient samples, Gemoll et al. demonstrates [
158] that in, comparison to fetal osteoblast, CatD immunostaining localized in the cytoplasm is overexpressed in OS and pulmonary metastases. Prognostically, CatD reached a sensitivity of 76.47% at 100% specificity, as well as a sensitivity of 100% at 100% specificity to predict OS and pulmonary metastasis, respectively [
158]. In vitro, Spreafico et al. [
159] observed that CatD is also upregulated in human OS cell line Saos-2 in comparison to mature osteoblasts (
Figure 2). The mRNA and protein expression of Saos-2 and its metastatic sublines LM5 and LM7 demonstrate a 2.5-fold upregulation of CatD [
55]. Contrarily, Arkona et al., demonstrates that CatD is not elevated in bone metastases [
156].
Elevated CatD in BCa tissue has been correlated to poor prognoses, and is a strong prognostic marker in BCa [
116,
160,
161,
162,
163]. CatD expression is also associated with the expression of CatG, L in BCa [
116]. Using BM2 to detect tumor-associated glycoprotein TAG12, which is expressed by almost all BCa cells and the anti-CatD antibody, Solomayer et al. demonstrated that patients with CatD-positive cells in their bone marrow have a significantly shorter metastasis-free interval (38 months) compared to patients who are CatD negative (64.5 months) [
164]. The CatD present in patient bone marrow was only found on tumor cells, and was not found on stromal cells in the bone marrow of patients with primary BCa [
164], suggesting that CatD may potentially serve as a clinical biomarker for bone malignancies to further guide treatment regimens.
The secretion of cathepsins into the microenvironment may also contribute to therapeutic drug resistance [
165] For example, secreted CatD attenuates apoptosis through the P13-Akt pathway, which leads to chemoresistance [
166]. CatD production also is implicated in doxorubicin (dox) resistance in OS and BCa [
165,
167].
The presence of CatG within the tumor has been demonstrated to increase metastasis [
168]. CatG is associated with the expression of cysteine proteases CatK and CatL in BCa patients [
116]. CatG is up-regulated at the tumor–bone interface and increases osteoclast differentiation, thereby inducing osteolytic lesions [
169]. CatG also activates pro-MMP9; the activated MMP9 in turn cleaves and releases active TGF- β, which promotes tumor growth and activated osteoclast and bone resorption [
143]. Wilson et al. [
143] also demonstrated that the inhibition of CatG in vivo via N-tosyl-l-phenylalanine chloromethyl ketone (TPCK; 50 mg/kg/d subcutaneous) significantly decreases MMP9 activity and reduces TGF- β signaling at the tumor–bone interface using the C166 adenocarcinoma cell line in BALB/c mice.
Rojinik et al. [
56] reported that CatH regulates bone morphogenetic protein 4 (BMP-4) in mice, but not in human OS and PCa. Previous studies demonstrated that CatH and BMP-4 expression is related to lung branching in mice, and that the inhibition of CatH leads to an accumulation of BMP-4 in mouse lungs [
170]. Contrary to the human OS cell line HOS, which does not express mature CatH, the PCa cell line PC-3 does expresses mature CatH [
56]. The addition of mature CatH into the culture with HOS increased the mRNA expression of BMP-4 2-fold, and decreased BMP-4 mRNA levels 0.5-fold in PC-3. Confirming Rojnik et al.’s studies, Husmann et al., also showed that mature CatH is undetectable in the Saos-2 parental and metastatic sublines (LM5 and LM7) [
55]. In OS, CatH does not appear to be correlated to metastasis because the protein expression of LM5 and LM7 are downregulated compared to Saos-2 [
55]. CatH appears to influence the relative expression of the BMP family members by upregulating the mRNA expression of BMP-3, -6, and -7, and down regulating BMP-1, -4, -5, and -8. However, Rojnik et al. [
56] demonstrated that, in the human OS (HOS) and PCa (PC-3) cell lines, CatH and BMP-4 do not co-localize, and the inhibition of CatH does not increase the expression of BMP-4 and has no direct impact on the processing or degradation of BMP-4. These findings suggest that CatH may indirectly regulate BMP-4, although the mechanism has not been elucidated [
56].
CatK is overexpressed in human cancer, and is associated with primary tumor growth and the metastatic process [
55,
171]. The development of primary bone tumors, such as OS, is associated with a local enhancement of CatK expression and excretion resulting in pathological excessive bone resorption [
78]. The CatK in primary and metastatic tumors is involved in tumor cell invasion via ECM degradation, osteolysis, and the modulation of cytokines and chemokines such as IL-1α and CCL2 [
171]. In 63% of OS tissue samples, tumor-associated osteoclasts were found within the tumor mass [
168]. The presence of osteoclasts in OS biopsies at diagnosis correlated with metastases in 50% of clinical cases, whereas 100% of patients with no osteoclasts present within the primary tumor did not have clinically-detectable metastases at diagnosis [
168]. In 90% of OS cases with osteoclast infiltration, CatK mRNA was present [
168]. Serum tartrate-resistant acid phosphatase 5b (TRACP 5b), a marker of bone resorption, has been significantly correlated to serum N-terminal telopeptide (NTx), a biomarker of bone turnover, and CatK mRNA in tumor tissues [
168]. Neoplastic cells expressing CatK and multinucleated tartrate-resistant acid phosphatase (TRAcP) have direct contact with the bone trabeculae and bone marrow spaces near metastatic tumor cells [
172]. TRACP 5b has been demonstrated to correlate with tumors’ aggressiveness in OS [
168].
The mRNA and protein expression of Saos-2 and its metastatic sublines LM5 and LM7 demonstrate a 2-fold upregulation of CatK [
55]. The osteoclast differentiation induced from co-cultured human OS cell line MG-63 with human macrophages results in the production of cytokines such as RANKL and colony stimulating factor-1 (CSF-1) [
110]. CatK demonstrates activity at the tumor–bone interface, with no CatK levels detected away from the bone [
124]. While it is absent in cells, the CatK activity on bone substrate increases several-fold during multinucleated differentiation [
110]. No NF-k B sites occur near the CatK transcription site [
173,
174,
175]; therefore, the specific osteoclast products involve unidentified transcription factors [
110].
CatK has also been implicated in metastatic OS [
55]. Approximately 15% of high grade non-metastatic OS patients and 53% of patients with high grade metastatic OS stained intensity for CatK [
55]. Husmann et al. demonstrated that, in patients with metastatic high-grade OS and low CatK expression at the time of diagnosis, the survival was significantly better than in those patients with high CatK staining, demonstrating that CatK expression may be of predictive prognostic value for patients with high-grade metastatic tumors at diagnosis [
55].
Leung et al., detected high CatK expression by immunohistochemistry in breast (45%, n = 88) and prostate (75%, n = 64) tumors [
176]. CatK mRNA increased approximately 3-fold in metastatic tissue compared to primary tumor tissue [
107]. In comparison to primary BCa and PCa samples, CatK is upregulated in bone metastases, and it is associated with increased tumor cell invasiveness [
58,
109].
In BCa, there is a statistical difference between healthy sex-matched controls and patients with primary or metastatic BCa [
144]. Tumminello et al. reported that, although CatK serum levels are decreased (mean 1.2–1.9 pmol/L) in BCa patients in comparison to their sex-matched healthy controls (mean: 11.3–14.0 pmol/L), cystatin C levels were significantly higher in BCa patients (mean: 1.5–0.59 mg/L) than their controls (mean: 0.85–0.16 mg/L) [
144]. Although cystatin C is a useful clinical marker for the differentiation of cancer patients from healthy controls, the cystatin C serum levels in primary and metastatic BCa and PCa patients are not significantly different [
144].
CatK is positive in 12% of PCa tumor cell samples and 31% of stroma cells surrounding the tumor [
177], but whereas cystatin C serum levels are significantly increased in PCa patients (1.7–0.65 mg/L), serum CatK levels are not [
144]. The authors also noted that, in PCa, there is no statistical difference between the controls and cancer patients who have primary or metastatic lesions to the bone. Intense immunoreactivity for CatK was observed in osteoclasts, and elevated levels of serum NTx were detected in patients with bone metastases [
109]. The administration of ZOL to patients with bone metastasis induces a significant increase of CatK and cystatin C serum levels in PCa patients [
144]. These findings suggest that cystatin C may be regarded as a possible marker to monitor the therapeutic response to bisphosphonate treatment (i.e: ZOL). The understanding of the role of CatK in the bone microenvironment has led to a multitude of efforts to evaluate whether CatK inhibition is an effective therapeutic strategy for patients with BCa or PCa that metastasizes to the bone.
CatK secretion from PCa cell lines (LNCaP, DU145) and macrophages participates in local invasion by mediating extracellular degradation [
109,
177]. Liang et al. [
178] reported that CatK expression occurs both in PCa cell lines (LNCaP, C4-2B, and PC3) and in PCa tissues. CatK protein and enzymatic activity has been detected in the human PCa cell lines by Western blot and a fluorogenic assay, respectively [
109].
Tumor-bearing osteoclasts secrete large amounts of cysteine proteases, especially pro-CatL, leading to tumor-associated bone absorption i.e., bone pit formations and the release of bone calcium, [
179]. Typically, CatL expression and secretion is associated with pro tumorigenic factors and osteoclastic cytokines, such as (IL-1, IL-6, and TNF-α), PTH, vascular endothelial growth factor (VEGF), and cellular-SRC [
116,
135,
180,
181,
182,
183].
Damiens et al. showed that, in human OS cell lines, MG-63 and Saos2 CatL activity was increased in the presence of IL-1, IL-6 and oncostatin M (hOSM), which is a cytokine of the IL-6 family. hOSM induces osteoblast cell proliferation, matrix protein synthesis, and IL-6 secretion in osteoblasts [
184,
185]. Human growth factor and IGF-1 decrease cathepsin activity in OS media. In human OS, there is a prevalence of CatL activity over CatB activity using the CatB and L substrate Z-Phe-Arg-AMC [
186]. The 2-fold elevation of Z-Phe-Arg-AMC hydrolysis in comparison to fibroblasts in metastatic bone cancer suggests that CatL has an active function in the metastatic process [
156]. These results differ from the findings of Aisa et al. [
43], who demonstrated a prevalence of CatB activity using the CatB-specific substrate Z-Arg-Arg-AMC.
In 50% of primary bone tumor tissue and 100% of metastatic tumor samples, CatL mRNA is expressed [
187]. Arkona et al. [
156] observed an elevation of CatL, but not other cysteine cathepsins such as Cat D and CatH, suggesting a possibly unique role of CatL in metastasis. In vitro studies in OS indicate that CatL contributes to the metastatic potential of neoplastic cells [
55,
187]. The mRNA and protein expression of Saos-2 and its metastatic sublines LM5 and LM7 demonstrate an upregulation of CatL in metastasis; however, the mature active form of CatL increases in the metastatic OS cell line LM5, but not in the parental Saos-2 or metastatic subline LM7 [
55].
The secretion of CatL in the microenvironment may also contribute to therapeutic drug resistance. CatL was associated with poor response to neoadjuvant chemotherapy, and CatL-positive patients achieved a lower complete remission rate compared to CatL-negative patients [
108]. However, CatL does not influence event-free-survival (EFS) or overall survival (OS) [
108].