Abstract
IntroductionInflammatory breast cancer (IBC) is an aggressive, metastatic and highly angiogenic form of locally advanced breast cancer with a relatively poor three-year survival rate. Breast cancer invasion has been linked to proteolytic activity at the tumor cell surface. Here we explored a role for active cathepsin B on the cell surface in the invasiveness of IBC.MethodsWe examined expression of the cysteine protease cathepsin B and the serine protease urokinase plasminogen activator (uPA), its receptor uPAR and caveolin-1 in two IBC cell lines: SUM149 and SUM190. We utilized a live cell proteolysis assay to localize in real time the degradation of type IV collagen by IBC cells. IBC patient biopsies were examined for expression of cathepsin B and caveolin-1.ResultsBoth cell lines expressed comparable levels of cathepsin B and uPA. In contrast, levels of caveolin-1 and uPAR were greater in SUM149 cells. We observed that uPA, uPAR and enzymatically active cathepsin B were colocalized in caveolae fractions isolated from SUM149 cells. Using a live-cell proteolysis assay, we demonstrated that both IBC cell lines degrade type IV collagen. The SUM149 cells exhibit predominantly pericellular proteolysis, consistent with localization of proteolytic pathway constitutents to caveolar membrane microdomains. A functional role for cathepsin B was confirmed by the ability of CA074, a cell impermeable and highly selective cathepsin B inhibitor, to significantly reduce pericellular proteolysis and invasion by SUM149 cells. A statistically significant co-expression of cathepsin B and caveolin-1 was found in IBC patient biopsies, thus validating our in vitro data.ConclusionOur study is the first to show that the proteolytic activity of cathepsin B and its co-expression with caveolin-1 contributes to the aggressiveness of IBC.
Highlights
Inflammatory breast cancer (IBC) is an aggressive, metastatic and highly angiogenic form of locally advanced breast cancer with a relatively poor three-year survival rate
A 38 kDa intermediate form of the enzyme was present only in lysates of the SUM149 cells and mature double chain cathepsin B was secreted only from SUM190 cells (Figure 1a); these differences between the two cell lines are likely to be due to variations in the extent of processing of the proenzyme into the various mature forms, as we have observed in a variety of human tumor tissues [28]
UPAR, caveolin-1 and b1-integrin expression is higher in SUM149 than in SUM190 cells The cell surface receptor for urokinase plasminogen activator (uPA) is urokinase receptor (uPAR) [32,33], a GPI-anchored membrane glycoprotein that has been localized to caveolae in a wide variety of cells [12,23,24]
Summary
Inflammatory breast cancer (IBC) is an aggressive, metastatic and highly angiogenic form of locally advanced breast cancer with a relatively poor three-year survival rate. We explored a role for active cathepsin B on the cell surface in the invasiveness of IBC. In 2010, an international panel of leading experts recommended the clinical consensus for a minimal standard diagnosis of IBC include erythema occupying at least one-third of the breast, hardening and retraction of the nipple, dimpling (peau d’orange) of the skin, and no response to antibiotic treatment [2]. These clinical characteristics are accompanied by extensive dermal lymphovascular. Downregulation of caveolin-1, the structural protein of caveolae, reduces the cell surface association of cathepsin B and decreases degradation of type IV collagen and invasion by the colon cancer cells, consistent with a functional role for caveolae-associated cathepsin B in invasion [12]
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