Article

Cathepsin B Inhibition Limits Bone Metastasis in Breast Cancer

Research Division, Peter MacCallum Cancer Centre, East Melbourne, Australia.
Cancer Research (Impact Factor: 9.28). 03/2012; 72(5):1199-209. DOI: 10.1158/0008-5472.CAN-11-2759
Source: PubMed

ABSTRACT Metastasis to bone is a major cause of morbidity in breast cancer patients, emphasizing the importance of identifying molecular drivers of bone metastasis for new therapeutic targets. The endogenous cysteine cathepsin inhibitor stefin A is a suppressor of breast cancer metastasis to bone that is coexpressed with cathepsin B in bone metastases. In this study, we used the immunocompetent 4T1.2 model of breast cancer which exhibits spontaneous bone metastasis to evaluate the function and therapeutic targeting potential of cathepsin B in this setting of advanced disease. Cathepsin B abundancy in the model mimicked human disease, both at the level of primary tumors and matched spinal metastases. RNA interference-mediated knockdown of cathepsin B in tumor cells reduced collagen I degradation in vitro and bone metastasis in vivo. Similarly, intraperitoneal administration of the highly selective cathepsin B inhibitor CA-074 reduced metastasis in tumor-bearing animals, a reduction that was not reproduced by the broad spectrum cysteine cathepsin inhibitor JPM-OEt. Notably, metastasis suppression by CA-074 was maintained in a late treatment setting, pointing to a role in metastatic outgrowth. Together, our findings established a prometastatic role for cathepsin B in distant metastasis and illustrated the therapeutic benefits of its selective inhibition in vivo.

1 Follower
 · 
205 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: Nitrile-type inhibitors are known to interact with cysteine proteases in a covalent-reversible manner. The chemotype of 3-cyano-3-aza-β-amino acid derivatives was designed in which the N-cyano group is centrally arranged in the molecule to allow for interactions with the nonprimed and primed binding regions of the target enzymes. These compounds were evaluated as inhibitors of the human cysteine cathepsins K, S, B, and L. They exhibited slow-binding behavior and were found to be exceptionally potent, in particular toward cathepsin K, with second-order rate constants up to 52 900 × 10(3) M(-1) s(-1).
    ACS Medicinal Chemistry Letters 10/2014; 5(10):1076-81. DOI:10.1021/ml500238q · 3.07 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The vacuolar (H+)-ATPases (V-ATPases) are a family of ATP-driven proton pumps that couple ATP hydrolysis with translocation of protons across membranes. Previous studies have implicated V-ATPases in cancer cell invasion. It has been proposed that V-ATPases participate in invasion by localizing to the plasma membrane and causing an acidification of the extracellular space. To test this hypothesis, we utilized two separate approaches to specifically inhibit plasma membrane V-ATPases. First, we stably transfected highly invasive MDA-MB231 cells with a V5-tagged construct of the membrane-embedded c subunit of the V-ATPase, allowing for extracellular expression of the V5 epitope. We evaluated the effect of addition of a monoclonal antibody directed against the V5 epitope on both V-ATPase-mediated proton translocation across the plasma membrane and invasion using an in vitro Matrigel assay. Addition of anti-V5 antibody resulted in acidification of the cytosol and a decrease in V-ATPase-dependent proton flux across the plasma membrane in transfected but not control (untransfected) cells. These results demonstrate that the anti-V5 antibody inhibits activity of plasma membrane V-ATPases in transfected cells. Addition of the anti-V5 antibody also inhibited in vitro invasion of transfected (but not untransfected) cells. Second, we utilized a biotin-conjugated form of the specific V-ATPase inhibitor bafilomycin. When bound to streptavidin, this compound cannot cross the plasma membrane. Addition of this compound to MDA-MB231 cells also inhibited in vitro invasion. These studies suggest that plasma membrane V-ATPases play an important role in invasion of breast cancer cells. Copyright © 2014, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 12/2014; 290(6). DOI:10.1074/jbc.M114.611210 · 4.60 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Macrophages play important roles in many diseases and are frequently found in hypoxic areas. A chronic hypoxic microenvironment alters global cellular protein expression but molecular details remain poorly understood. Although hypoxia inducible factor (HIF) is an established transcription factor allowing adaption to acute hypoxia, responses to chronic hypoxia are more complex. Based on a two-dimensional differential gel electrophoresis (2D-DIGE) approach, we aimed to identify proteins that are exclusively expressed under chronic but not acute hypoxia (1% O2). One of the identified proteins was cathepsin B (CTSB) and a knockdown of either HIF-1α or -2α in primary human macrophages pointed to a HIF-2α-dependency. Although ChIP experiments confirmed HIF-2 binding to a CTSB enhancer in acute hypoxia, an increase of CTSB mRNA was only evident under chronic hypoxia. Along those lines, CTSB mRNA stability increased at 48 h but not at 8 h of hypoxia. However, RNA stability at 8 h hypoxia was enhanced by a knockdown of tristetraprolin (TTP). Inactivation of TTP under prolonged hypoxia was facilitated by c-jun N-terminal kinase (JNK) and inhibition of this kinase lowered CTSB mRNA levels and stability. We postulate a TTP-dependent mechanism to explain delayed expression of CTSB under chronic hypoxia. Copyright © 2014, American Society for Microbiology. All Rights Reserved.
    Molecular and Cellular Biology 12/2014; 35(3). DOI:10.1128/MCB.01034-14 · 5.04 Impact Factor