Abstract
BackgroundNormal epithelial cells and carcinoma cells can acquire invasiveness by epithelial-to-mesenchymal transition (EMT), a process of considerable cellular remodeling. The endosomal/lysosomal compartment is a principal site of intracellular protein degradation. Lysosomal cathepsin proteases are secreted during cancer progression. The established pro-metastatic role of specific cysteine cathepsins has until now been ascribed to their contribution to extracellular matrix remodeling. We hypothesized that cysteine cathepsins affect transforming growth factor β-1 (TGFβ-1)-induced EMT of normal and malignant mammary epithelial cells.MethodsThe role of lysosomal proteolysis in TGFβ-1-induced EMT and invasion was investigated in a normal and a novel malignant murine mammary epithelial cell line. The contribution of cysteine cathepsins was determined by addition of the general cysteine cathepsin inhibitor E64d. Hallmarks of EMT were analyzed by molecular- and cell-biologic analyses including real-time cell migration/invasion assays. A quantitative proteome comparison using stable isotopic labeling with amino acids in culture (SILAC) showed the effect of E64d on TGFβ-1 induced proteome changes. Lysosomal patterning and junctional adhesion molecule A (Jam-a) localization and abundance were analyzed by immunofluorescence.ResultsWe found increased lysosome activity during EMT of malignant mammary epithelial cells. Cysteine cathepsin inhibition had no effect on the induction of the TGFβ-1-induced EMT program on transcriptional level. Protease inhibition did not affect invasion of TGFβ-1 treated normal mammary epithelial cells, but reduced the invasion of murine breast cancer cells. Remarkably, reduced invasion was also evident if E64d was removed 24 h before the invasion assay in order to allow for recovery of cathepsin activity. Proteome analyses revealed a high abundance of lysosomal enzymes and lysosome-associated proteins in cancer cells treated with TGFβ-1 and E64d. An accumulation of those proteins and of lysosomal vesicles was further confirmed by independent methods. Interestingly, E64d caused lysosomal accumulation of Jam-a, a tight junction component facilitating epithelial cell-cell adhesion.ConclusionOur results demonstrate an important role of lysosomal proteolysis in cellular remodeling during EMT and a pivotal contribution of lysosomal cysteine cathepsins to TGFβ-1 induced acquisition of breast cancer cell invasiveness. These findings provide an additional rationale to use cathepsin inhibitors to stall tumor metastasis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0313-5) contains supplementary material, which is available to authorized users.
Highlights
Metastatic spread of breast cancers is responsible for most breast cancer deaths
epithelial-to-mesenchymal transition (EMT) plasticity and cathepsin expression in normal and malignant mammary epithelial cells in response to Transforming growth factor beta (TGFβ)-1 Cysteine cathepsin functions during transforming growth factor β-1 (TGFβ-1) induced EMT were analyzed using normal murine mammary gland epithelial cells (NMuMG) cells [21] and a novel MMTV-PyMT breast cancer cell line “Immortalized polyoma-luciferase 32 (iPL32)”, respectively. iPL32 cells were established from primary tumors of a 14 week old MMTVPyMT-Luctg mouse
The active forms of cysteine cathepsins cathepsin L (Ctsl), Ctsc, and Ctsh increased during four days of TGFβ-1 treatment in NMuMG cells
Summary
Metastatic spread of breast cancers is responsible for most breast cancer deaths. The first critical step of cancer cells leaving a solid tumor is the loss of epithelial integrity and the gain of migratory and invasive capabilities. The complex canonical and non-canonical intracellular TGFβ-1 signal transduction is modified by ligand-induced endocytosis of monoubiquitinylated TGFβ-receptor/ligand complexes [4] At this point TGFβ-1 signaling meets the endolysosomal compartment (hereafter referred to as lysosomes), which represents the site for processing and degradation of proteins delivered by endocytic and autophagic pathways [5,6]. Cathepsins can be secreted and their tumorigenic and pro-metastatic functions have been mainly ascribed to their ability to directly degrade extracellular matrix (ECM) proteins or activate an extracellular proteolytic cascade [15,16]. In contrast their association with lysosomemediated cell death pathways implies an anti-tumorigenic role [17]. We hypothesized that cysteine cathepsins affect transforming growth factor β-1 (TGFβ-1)-induced EMT of normal and malignant mammary epithelial cells
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