Purpose: Cathepsin S is a cysteine protease that promotes the invasion of tumor and endothelial cells during cancer progression. Here we investigated the potential to target cathepsin S using an antagonistic antibody, Fsn0503, to block these tumorigenic effects. Experimental Design: A panel of monoclonal antibodies was raised to human cathepsin S. The effects of a selected antibody were subsequently determined using invasion and proteolysis assays. Endothelial cell tube formation and aorta sprouting assays were done to examine antiangiogenic effects. In vivo effects were also evaluated using HCT116 xenograft studies. Results: A selected cathepsin S antibody, Fsn0503, significantly blocked invasion of a range of tumor cell lines, most significantly HCT116 colorectal carcinoma cells, through inhibition of extracellular cathepsin S-mediated proteolysis. We subsequently found enhanced expression of cathepsin S in colorectal adenocarcinoma biopsies when compared with normal colon tissue. Moreover, Fsn0503 blocked endothelial cell capillary tube formation and aortic microvascular sprouting. We further showed that administration of Fsn0503 resulted in inhibition of tumor growth and neovascularization of HCT116 xenograft tumors. The lysosomal cysteine cathepsins encompass a family of closely related cysteine proteases, mediating a diverse range of proteolytic effects (1-4). However, an increasing body of evidence has shown the overexpression of a number of cysteine cathepsins in cancer (5-7). Significantly, these proteases are secreted into the tumor extracellular milieu, producing potent degradative effects on a broad range of extracellular matrix (ECM) components, including collagen and laminins (8-10). Further confirmation of these effects were provided in a murine model of sporadic pancreatic carcinogenesis (RIP1-Tag2), in which the genetic ablation of either cathepsin B or cathepsin S severally attenuated tumor invasion and angiogenesis, and cathepsin L or cathepsin B deficiency inhibited tumor proliferation (11). These observations highlight their potential as therapeutic targets in cancer treatment. Indeed, the application of synthetic broad-spectrum probes and combination therapies has successfully shown efficacy in vivo using various tumor models (12-15). However, given the roles that these proteases play in normal cellular homeostasis, an approach that selectively targets a specific cathepsin with limited normal tissue distribution may be more therapeutically attractive.Cathepsin S, unlike the ubiquitous cathepsin B and cathepsin L, exhibits a restricted tissue expression. It is found predominantly in lymphatic tissue, macrophages, and other professional antigenpresenting cells (16); mediating key steps in antigen presentation through cleavage of the invariant chain (17,18). However, the inappropriate expression of cathepsin S has also been observed in a range of tumors such as astrocytomas (19-21), prostate (22), hepatocellular (23), and pancreatic carcinomas (11). Crucially, evidence from the RIP1-T...
Androgen withdrawal induces hypoxia in androgen-sensitive tissue; this is important as in the tumour microenvironment, hypoxia is known to drive malignant progression. Our study examined the time-dependent effect of androgen deprivation therapy (ADT) on tumour oxygenation and investigated the role of ADT-induced hypoxia on malignant progression in prostate tumours. LNCaP xenografted tumours were treated with anti-androgens and tumour oxygenation measured. Dorsal skin fold (DSF) chambers were used to image tumour vasculature in vivo. Quantitative PCR (QPCR) identified differential gene expression following treatment with bicalutamide. Bicalutamide-treated and vehicle-only-treated tumours were re-established in vitro, and invasion and sensitivity to docetaxel were measured. Tumour growth delay was calculated following treatment with bicalutamide combined with the bioreductive drug AQ4N. Tumour oxygenation measurements showed a precipitate decrease following initiation of ADT. A clinically relevant dose of bicalutamide (2 mg/kg/day) decreased tumour oxygenation by 45% within 24 hr, reaching a nadir of 0.09% oxygen (0.67 6 0.06 mmHg) by Day 7; this persisted until Day 14 when it increased up to Day 28. Using DSF chambers, LNCaP tumours treated with bicalutamide showed loss of small vessels at Days 7 and 14 with revascularisation occurring by Day 21. QPCR showed changes in gene expression consistent with the vascular changes and malignant progression. Cells from bicalutamide-treated tumours were more malignant than vehicle-treated controls. Combining bicalutamide with AQ4N (50 mg/kg, single dose) caused greater tumour growth delay than bicalutamide alone. Our study shows that bicalutamide-induced hypoxia selects for cells that show malignant progression; targeting hypoxic cells may provide greater clinical benefit.
BackgroundAngiogenesis is a key hallmark of tumourigenesis and its inhibition is a proven strategy for the development of novel anti-cancer therapeutics. An important aspect of early angiogenesis is the co-ordinated migration and invasion of endothelial cells through the hypoxic tumour tissue. Cathepsin S has been shown to play an important role in angiogenesis as has vascular endothelial growth factor (VEGF). We sought to assess the anti-angiogenic effect of Fsn0503, a novel cathepsin S inhibitory antibody, when combined with anti-VEGF on vascular development.Methodology/Principal FindingsCathepsin S expression and secretion from endothelial cells was characterised using RT-PCR and western blotting. We further show that cathepsin S promotes pericellular hydrolysis of extracellular matrix components in the tumour microenvironment and facilitates endothelial invasion. The cathepsin S inhibitory antibody, Fsn0503, blocks extracellular proteolysis, inhibiting endothelial invasion and tube formation in cell-based assays. The anti-angiogenic effects of Fsn0503 were also shown in vivo where it significantly retarded the development of vasculature in human xenograft models. Furthermore, when Fsn0503 was combined with an anti-VEGF antibody, a synergistic inhibition of microvascular development was observed.Conclusions/SignificanceTaken together, this data demonstrates that the antibody-mediated targeting of cathepsin S represents a novel method of inhibiting angiogenesis. Furthermore, when used in combination with anti-VEGF therapies, Fsn0503 has the potential to significantly enhance current treatments of tumour neovascularisation and may also be of use in the treatment of other conditions associated with inappropriate angiogenesis.
Drug metabolizing transgene products, which activate bioreductive cytotoxins, can be used to target treatment-resistant hypoxic tumors. The prodrug AQ4N is bioreduced in hypoxic cells by cytochrome P450s (CYPs) to the cytotoxin AQ4. Previously we have shown that intra-tumoral injection of CYP3A4 and CYP2B6 transgenes with AQ4N and radiation inhibits tumor growth. Here we examine the ability of other CYPs, in particular CYP1A1, to metabolize AQ4N, and to enhance radiosensitization. Metabolism of AQ4N was assessed using microsomes prepared from baculovirus-infected cells transfected with various CYP isoforms. AQ4N metabolism was most efficient with CYP1A1 (66.7 nmol/min/pmol) and 2B6 (34.4 nmol/min/pmol). Transient transfection of human CYP1A17CYP reductase (CYPRED) was investigated in hypoxic RIF-1 mouse cells in vitro using the alkaline comet assay. There was a significant increase in DNA damage following transient transfection of CYP1A1 compared to non-transfected cells; inclusion of CYPRED provided no additional effect. In vivo, a single intra-tumoral injection of a CYP1A1 construct in combination with AQ4N (100 mg/kg i.p.) and 20 Gy X-rays caused a 16-day delay in tumor regrowth compared to tumors receiving AQ4N plus radiation and empty vector (P ¼ 0.0344). The results show the efficacy of a CYP1A1-mediated GDEPT strategy for bioreduction of AQ4N.
BackgroundProteolytic enzymes have been implicated in driving tumor progression by means of their cancer cell microenvironment activity where they promote proliferation, differentiation, apoptosis, migration, and invasion. Therapeutic strategies have focused on attenuating their activity using small molecule inhibitors, but the association of proteases with the cell surface during cancer progression opens up the possibility of targeting these using antibody dependent cellular cytotoxicity (ADCC). Cathepsin S is a lysosomal cysteine protease that promotes the growth and invasion of tumour and endothelial cells during cancer progression. Our analysis of colorectal cancer patient biopsies shows that cathepsin S associates with the cell membrane indicating a potential for ADCC targeting.ResultsHere we report the cell surface characterization of cathepsin S and the development of a humanized antibody (Fsn0503h) with immune effector function and a stable in vivo half-life of 274 hours. Cathepsin S is expressed on the surface of tumor cells representative of colorectal and pancreatic cancer (23%-79% positive expression). Furthermore the binding of Fsn0503h to surface associated cathepsin S results in natural killer (NK) cell targeted tumor killing. In a colorectal cancer model Fsn0503h elicits a 22% cytotoxic effect.ConclusionsThis data highlights the potential to target cell surface associated enzymes, such as cathepsin S, as therapeutic targets using antibodies capable of elicitingADCC in tumor cells.
Purpose: A number of cytotoxic chemotherapy agents tested at low concentrations show antiangiogenic properties with limited cytotoxicity, e.g., cyclophosphamide, tirapazamine, and mitoxantrone. AQ4N is a bioreductive alkylaminoanthraquinone that is cytotoxic when reduced to AQ4; hence, it can be used to target hypoxic tumor cells. AQ4N is structurally similar to mitoxantrone and was evaluated for antiangiogenic properties without the need for bioreduction. Experimental Design: The effect of AQ4N and fumagillin on human microvascular endothelial cells (HMEC-1) was measured using a variety of in vitro assays, i.e., 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, wound scrape, tubule formation, rat aortic ring, and invasion assays. Low-dose AQ4N (20 mg/kg) was also given in vivo to mice bearing a tumor in a dorsal skin flap. Results: AQ4N (10-5 mol/L) had no effect on HMEC-1viability. AQ4N (10 -9 to10-5 mol/L) caused a sigmoidal dose-dependent inhibition of endothelial cell migration in the wound scrape model. Fumagillin showed a similar response over a lower dose range (10 -13 to 10 -9 mol/L); however, the maximal inhibition was less (25% versus 43% for AQ4N). AQ4N inhibited HMEC-1 cell contacts on Matrigel (10 -8 to 10 -5 mol/L), HMEC-1 cell invasion, and sprouting in rat aorta explants. Immunofluorescence staining with tubulin, vimentim, dynein, and phalloidin revealed that AQ4N caused disruption to the cell cytoskeleton. When AQ4N (20 mg/kg) was given in vivo for 5 days, microvessels disappeared in LNCaP tumors grown in a dorsal skin flap. Conclusions: This combination of assays has shown that AQ4N possesses antiangiogenic effects in normoxic conditions, which could potentially contribute to antitumor activity.
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