A 9-kDa matricellular SPARC fragment released by cathepsin D exhibits pro-tumor activity in the triple-negative breast cancer microenvironment.

Rationale: Alternative therapeutic strategies based on tumor-specific molecular targets are urgently needed for triple-negative breast cancer (TNBC). The protease cathepsin D (cath-D) is a marker of poor prognosis in TNBC and a tumor-specific extracellular target for antibody-based therapy. The identification of cath-D substrates is crucial for the mechanistic understanding of its role in the TNBC microenvironment and future therapeutic developments. Methods: The cath-D substrate repertoire was investigated by N-Terminal Amine Isotopic Labeling of Substrates (TAILS)-based degradome analysis in a co-culture assay of TNBC cells and breast fibroblasts. Substrates were validated by amino-terminal oriented mass spectrometry of substrates (ATOMS). Cath-D and SPARC expression in TNBC was examined using an online transcriptomic survival analysis, tissue micro-arrays, TNBC cell lines, patient-derived xenografts (PDX), human TNBC samples, and mammary tumors from MMTV-PyMT Ctsd-/- knock-out mice. The biological role of SPARC and its fragments in TNBC were studied using immunohistochemistry and immunofluorescence analysis, gene expression knockdown, co-culture assays, western blot analysis, RT-quantitative PCR, adhesion assays, Transwell motility, trans-endothelial migration and invasion assays. Results: TAILS analysis showed that the matricellular protein SPARC is a substrate of extracellular cath-D. In vitro, cath-D induced limited proteolysis of SPARC C-terminal extracellular Ca2+ binding domain at acidic pH, leading to the production of SPARC fragments (34-, 27-, 16-, 9-, and 6-kDa). Similarly, cath-D secreted by TNBC cells cleaved fibroblast- and cancer cell-derived SPARC at the tumor pericellular acidic pH. SPARC cleavage also occurred in TNBC tumors. Among these fragments, only the 9-kDa SPARC fragment inhibited TNBC cell adhesion and spreading on fibronectin, and stimulated their migration, endothelial transmigration, and invasion. Conclusions: Our study establishes a novel crosstalk between proteases and matricellular proteins in the tumor microenvironment through limited SPARC proteolysis, revealing a novel targetable 9-kDa bioactive SPARC fragment for new TNBC treatments. Our study will pave the way for the development of strategies for targeting bioactive fragments from matricellular proteins in TNBC.

Immunotherapy of triple-negative breast cancer with cathepsin D-targeting antibodies.

BACKGROUND: Triple-negative breast cancer (TNBC) treatment is currently restricted to chemotherapy. Hence, tumor-specific molecular targets and/or alternative therapeutic strategies for TNBC are urgently needed. Immunotherapy is emerging as an exciting treatment option for TNBC patients. The aspartic protease cathepsin D (cath-D), a marker of poor prognosis in breast cancer (BC), is overproduced and hypersecreted by human BC cells. This study explores whether cath-D is a tumor cell-associated extracellular biomarker and a potent target for antibody-based therapy in TNBC. METHODS: Cath-D prognostic value and localization was evaluated by transcriptomics, proteomics and immunohistochemistry in TNBC. First-in-class anti-cath-D human scFv fragments binding to both human and mouse cath-D were generated using phage display and cloned in the human IgG1 λ format (F1 and E2). Anti-cath-D antibody biodistribution, antitumor efficacy and in vivo underlying mechanisms were investigated in TNBC MDA-MB-231 tumor xenografts in nude mice. Antitumor effect was further assessed in TNBC patient-derived xenografts (PDXs). RESULTS: High CTSD mRNA levels correlated with shorter recurrence-free survival in TNBC, and extracellular cath-D was detected in the tumor microenvironment, but not in matched normal breast stroma. Anti-cath-D F1 and E2 antibodies accumulated in TNBC MDA-MB-231 tumor xenografts, inhibited tumor growth and improved mice survival without apparent toxicity. The Fc function of F1, the best antibody candidate, was essential for maximal tumor inhibition in the MDA-MB-231 model. Mechanistically, F1 antitumor response was triggered through natural killer cell activation via IL-15 upregulation, associated with granzyme B and perforin production, and the release of antitumor IFNγ cytokine. The F1 antibody also prevented the tumor recruitment of immunosuppressive tumor-associated macrophages M2 and myeloid-derived suppressor cells, a specific effect associated with a less immunosuppressive tumor microenvironment highlighted by TGFß decrease. Finally, the antibody F1 inhibited tumor growth of two TNBC PDXs, isolated from patients resistant or not to neo-adjuvant chemotherapy. CONCLUSION: Cath-D is a tumor-specific extracellular target in TNBC suitable for antibody-based therapy. Immunomodulatory antibody-based strategy against cath-D is a promising immunotherapy to treat patients with TNBC.

Nuclear cathepsin D enhances TRPS1 transcriptional repressor function to regulate cell cycle progression and transformation in human breast cancer cells.

The lysosomal protease cathepsin D (Cath-D) is overproduced in breast cancer cells (BCC) and supports tumor growth and metastasis formation. Here, we describe the mechanism whereby Cath-D is accumulated in the nucleus of ERα-positive (ER+) BCC. We identified TRPS1 (tricho-rhino-phalangeal-syndrome 1), a repressor of GATA-mediated transcription, and BAT3 (Scythe/BAG6), a nucleo-cytoplasmic shuttling chaperone protein, as new Cath-D-interacting nuclear proteins. Cath-D binds to BAT3 in ER+ BCC and they partially co-localize at the surface of lysosomes and in the nucleus. BAT3 silencing inhibits Cath-D accumulation in the nucleus, indicating that Cath-D nuclear targeting is controlled by BAT3. Fully mature Cath-D also binds to full-length TRPS1 and they co-localize in the nucleus of ER+ BCC where they are associated with chromatin. Using the LexA-VP16 fusion co-activator reporter assay, we then show that Cath-D acts as a transcriptional repressor, independently of its catalytic activity. Moreover, microarray analysis of BCC in which Cath-D and/or TRPS1 expression were silenced indicated that Cath-D enhances TRPS1-mediated repression of several TRPS1-regulated genes implicated in carcinogenesis, including PTHrP, a canonical TRPS1 gene target. In addition, co-silencing of TRPS1 and Cath-D in BCC affects the transcription of cell cycle, proliferation and transformation genes, and impairs cell cycle progression and soft agar colony formation. These findings indicate that Cath-D acts as a nuclear transcriptional cofactor of TRPS1 to regulate ER+ BCC proliferation and transformation in a non-proteolytic manner.

Proteolysis of cystatin C by cathepsin D in the breast cancer microenvironment.

The aspartic protease cathepsin D, a poor prognostic indicator of breast cancer, is abundantly secreted as procathepsin D by human breast cancer cells and self-activates at low pH in vitro, giving rise to catalytically active cathepsin D. Due to a lower extracellular pH in tumor microenvironments compared to normal tissues, cathepsin D may cleave pathophysiological substrates contributing to cancer progression. Here, we show by yeast 2-hybrid and degradomics analyses that cystatin C, the most potent natural secreted inhibitor of cysteine cathepsins, both binds to and is a substrate of extracellular procathepsin D. The amount of cystatin C in the extracellular environment is reduced in the secretome of mouse embryonic fibroblasts stably transfected with human cathepsin D. Cathepsin D extensively cleaved cystatin C in vitro at low pH. Cathepsin D secreted by breast cancer cells also processed cystatin C at the pericellular pH of tumors and so enhancing extracellular proteolytic activity of cysteine cathepsins. Thus, tumor derived cathepsin D assists breast cancer progression by reducing cystatin C activity, which, in turn, enhances cysteine cathepsin proteolytic activity, revealing a new link between protease classes in the protease web.

Cathepsin-D, a key protease in breast cancer, is up-regulated in obese mouse and human adipose tissue, and controls adipogenesis.

The aspartic protease cathepsin-D (cath-D) is overexpressed by human epithelial breast cancer cells and is closely correlated with poor prognosis in breast cancer. The adipocyte is one of the most prominent cell types in the tumor-microenvironment of breast cancer, and clinical studies have shown that obesity increases the incidence of breast cancer. Here, we provide the first evidence that cath-D expression is up-regulated in adipose tissue from obese human beings, as well as in adipocytes from the obese C57BI6/J mouse. Cath-D expression is also increased during human and mouse adipocyte differentiation. We show that cath-D silencing in 3T3-F442A murine preadipocytes leads to lipid-depleted cells after adipogenesis induction, and inhibits of the expression of PPARγ, HSL and aP2 adipocyte differentiation markers. Altogether, our findings demonstrate the key role of cath-D in the control of adipogenesis, and suggest that cath-D may be a novel target in obesity.

Pro-cathepsin D interacts with the extracellular domain of the beta chain of LRP1 and promotes LRP1-dependent fibroblast outgrowth.

Interactions between cancer cells and fibroblasts are crucial in cancer progression. We have previously shown that the aspartic protease cathepsin D (cath-D), a marker of poor prognosis in breast cancer that is overexpressed and highly secreted by breast cancer cells, triggers mouse embryonic fibroblast outgrowth via a paracrine loop. Here, we show the requirement of secreted cath-D for human mammary fibroblast outgrowth using a three-dimensional co-culture assay with breast cancer cells that do or do not secrete pro-cath-D. Interestingly, proteolytically-inactive pro-cath-D remains mitogenic, indicating a mechanism involving protein-protein interaction. We identify the low-density lipoprotein (LDL) receptor-related protein-1, LRP1, as a novel binding partner for pro-cath-D in fibroblasts. Pro-cath-D binds to residues 349-394 of the ß chain of LRP1, and is the first ligand of the extracellular domain of LRP1ß to be identified. We show that pro-cath-D interacts with LRP1ß in cellulo. Interaction occurs at the cell surface, and overexpressed LRP1ß directs pro-cath-D to the lipid rafts. Our results reveal that the ability of secreted pro-cath-D to promote human mammary fibroblast outgrowth depends on LRP1 expression, suggesting that pro-cath-D-LRP1ß interaction plays a functional role in the outgrowth of fibroblasts. Overall, our findings strongly suggest that pro-cath-D secreted by epithelial cancer cells promotes fibroblast outgrowth in a paracrine LRP1-dependent manner in the breast tumor microenvironment.

Pathophysiological functions of cathepsin D: Targeting its catalytic activity versus its protein binding activity?

The lysosomal aspartic protease cathepsin D (cath-D) is overexpressed and hyper-secreted by epithelial breast cancer cells. This protease is an independent marker of poor prognosis in breast cancer as it is correlated with the incidence of clinical metastasis. In normal cells, cath-D is localized in intracellular vesicles (lysosomes and endosomes). In cancer cells, overexpressed cath-D accumulates in cells, where it may affect their degradative capacities, and the pro-enzyme is hyper-secreted in the tumor micro-environment. In addition, during apoptosis, lysosomal cath-D is released into the cytosol, where it may interact with and/or cleave pro-apoptotic, anti-apoptotic, or nuclear proteins. Several studies have shown that cath-D affects various different steps in tumor progression and metastasis. Cath-D stimulates cancer cell growth in an autocrine manner, and also cath-D plays a crucial paracrine role in the tumor micro-environment by stimulating fibroblast outgrowth and tumor angiogenesis. A mutant D231N-cath-D, which is devoid of catalytic activity, remained mitogenic, indicating an additional action of cath-D by protein-protein interaction. Targeting cath-D in cancer may require the use of inhibitors of its catalytic activity, but also the development of new tools to inhibit its protein binding functions. Thus, elucidation of the mechanism of action of cath-D is crucial if an appropriate strategy is to be developed to target this protease in cancer. The discovery of new physiological substrates of cath-D using proteomic approaches can be expected to generate new critical targets. The aim of this review is to describe the roles of the cath-D protease in cancer progression and metastasis, as well as its function in apoptosis, and to discuss how it can be targeted in cancer by inhibiting its proteolytic activity and/or its binding protein activity.

Processing of human cathepsin D is independent of its catalytic function and auto-activation: involvement of cathepsins L and B.

The current mechanism proposed for the processing and activation of the 52 kDa lysosomal aspartic protease cathepsin D (cath-D) is a combination of partial auto-activation generating a 51 kDa pseudo-cath-D, followed by enzyme-assisted maturation involving cysteine and/or aspartic proteases and yielding successively a 48 kDa intermediate and then 34 + 14 kDa cath-D mature species. Here we have investigated the in vivo processing of human cath-D in a cath-D-deficient fibroblast cell line in order to determine whether its maturation occurs through already active cath-D and/or other proteases. We demonstrate that cellular cath-D is processed in a manner independent of its catalytic function and that auto-activation is not a required step. Moreover, the cysteine protease inhibitor E-64 partially blocks processing, leading to accumulation of 52-48 kDa cath-D intermediates. Furthermore, two inhibitors, CLICK148 and CA-074Met, specific for the lysosomal cath-L and cath-B cysteine proteases induce accumulation of 48 kDa intermediate cath-D. Finally, maturation of endocytosed pro-cath-D is also independent of its catalytic function and requires cysteine proteases. We therefore conclude that the mechanism of cath-D maturation involves a fully-assisted processing similar to that of pro-renin.

Cathepsin D: newly discovered functions of a long-standing aspartic protease in cancer and apoptosis.

The lysosomal aspartic protease cathepsin D (cath-D) is over-expressed and hyper-secreted by epithelial breast cancer cells. This protease is an independent marker of poor prognosis in breast cancer being correlated with the incidence of clinical metastasis. Cath-D over-expression stimulates tumorigenicity and metastasis. Indeed it plays an essential role in the multiple steps of tumor progression, in stimulating cancer cell proliferation, fibroblast outgrowth and angiogenesis, as well as in inhibiting tumor apoptosis. A mutated cath-D devoid of catalytic activity still proved mitogenic for cancer, endothelial and fibroblastic cells, suggesting an extra-cellular mode of action of cath-D involving a triggering, either directly or indirectly, of an as yet unidentified cell surface receptor. Cath-D is also a key mediator of induced-apoptosis and its proteolytic activity has been involved generally in this event. During apoptosis, mature lysosomal cath-D is translocated to the cytosol. Since cath-D is one of the lysosomal enzymes which requires a more acidic pH to be proteolytically-active relative to the cysteine lysosomal enzymes, such as cath-B and -L, it is open to question whether cytosolic cath-D might be able to cleave substrate(s) implicated in the apoptotic cascade. This review summarises our current knowledge on cath-D action in cancer progression and metastasis, as well as its dual function in apoptosis.

Down-regulation of cathepsin-D expression by antisense gene transfer inhibits tumor growth and experimental lung metastasis of human breast cancer cells.

Overexpression of cathepsin-D in primary breast cancer has been associated with rapid development of clinical metastasis. To investigate the role of this protease in breast cancer growth and progression to metastasis, we stably transfected a highly metastatic human breast cancer cell line, MDA-MB-231, with a plasmid containing either the full-length cDNA for cathepsin-D or a 535 bp antisense cathepsin-D cDNA fragment. Clones expressing antisense cathepsin-D cDNA that exhibited a 70-80% reduction in cathepsin-D protein, both intra- and extracellularly compared to controls, were selected for further experiments. These antisense-transfected cells displayed a reduced outgrowth rate when embedded in a Matrigel matrix, formed smaller colonies in soft agar and presented a significantly decreased tumor growth and experimental lung metastasis in nude mice compared with controls. However, manipulating the cathepsin-D level in the antisense cells has no effect on their in vitro invasiveness. These studies demonstrate that cathepsin-D enhances anchorage-independent cell proliferation and subsequently facilitates tumorigenesis and metastasis of breast cancer cells. Our overall results provide the first evidence on the essential role of cathepsin-D in breast cancer, and support the development of a new cathepsin-D-targeted therapy.