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The aspartyl-protease cathepsin D (cath-D) is overexpressed and hypersecreted by epithelial breast cancer cells and stimulates their proliferation. As tumor epithelial–fibroblast cell interactions are important events in cancer progression, we investigated whether cath-D overexpression affects also fibroblast behavior. We demonstrate a requirement of cath-D for fibroblast invasive growth using a three-dimensional (3D) coculture assay with cancer cells secreting or not pro-cath-D. Ectopic expression of cath-D in cath-D–deficient fibroblasts stimulates 3D outgrowth that is associated with a significant increase in fibroblast proliferation, survival, motility, and invasive capacity, accompanied by activation of the ras–MAPK pathway. Interestingly, all these stimulatory effects on fibroblasts are independent of cath-D proteolytic activity. Finally, we show that pro-cath-D secreted by cancer cells is captured by fibroblasts and partially mimics effects of transfected cath-D. We conclude that cath-D is crucial for fibroblast invasive outgrowth and could act as a key paracrine communicator between cancer and stromal cells, independently of its catalytic activity.

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.

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.

Autophagy is regulated by posttranslational modifications, including acetylation. Here we show that HLA-B-associated transcript 3 (BAT3) is essential for basal and starvation-induced autophagy in embryonic day 18.5 BAT3 −/− mouse embryos and in mouse embryonic fibroblasts (MEFs) through the modulation of p300-dependent acetylation of p53 and ATG7. Specifically, BAT3 increases p53 acetylation and proautophagic p53 target gene expression, while limiting p300-dependent acetylation of ATG7, a mechanism known to inhibit autophagy. In the absence of BAT3 or when BAT3 is located exclusively in the cytosol, autophagy is abrogated, ATG7 is hyperacetylated, p53 acetylation is abolished, and p300 accumulates in the cytosol, indicating that BAT3 regulates the nuclear localization of p300. In addition, the interaction between BAT3 and p300 is stronger in the cytosol than in the nucleus and, during starvation, the level of p300 decreases in the cytosol but increases in the nucleus only in the presence of BAT3. We conclude that BAT3 tightly controls autophagy by modulating p300 intracellular localization, affecting the accessibility of p300 to its substrates, p53 and ATG7.degradation | signalisation | nucleo-cytoplasmic shuttling A utophagy allows the lysosomal degradation of intracellular macromolecules and organelles after their sequestration in a vacuole known as the autophagosome (1). Basal autophagy is a cytoplasmic quality control mechanism that limits the production of reactive oxygen species and genomic instability. Autophagy is also induced to improve cell survival under stress.Signaling pathways involved in the regulation of autophagy have been widely studied (2). Autophagy is modulated at two levels: (i) the molecular machinery involved in autophagosome biogenesis, dependent on specific genes known as Atg (AuTophaGy) genes, and (ii) the upstream signaling pathways (e.g., PI3K, MAPK, mTOR) that act on ATG proteins. Posttranslational modifications are crucial for the regulation of autophagy. The first example came from Y. Oshumi's laboratory with the discovery of the conjugation systems for the ATG5-ATG12 and ATG8-phosphatidylethanolamine complexes in yeast (3). Phosphorylation is probably the most thoroughly investigated posttranslational event in autophagy. It appears that modulation of acetylation also affects Atg gene expression or activity; for instance, acetylation of the Unc-51-like kinase 1 (ULK1) (mammalian homolog of ATG1) by the acetylase Tat interacting protein 60 kDa (TIP60) induces autophagy after growth factor deprivation (4). In yeast, ESA1-dependent acetylation of ATG3 is essential for its interaction with ATG8 and ATG8 lipidation (5). Conversely, acetylation of ATG5, ATG7, microtubule associated protein 1 light chain 3 (LC3), and ATG12 by the acetyltransferase p300 inhibits autophagy (6), whereas their deacetylation by Sirtuins 1 (SIRT1) stimulates autophagy (7).HLA-B-associated transcript 3 (BAT3) is a nucleo-cytoplasmic shuttling protein that contains, among other, a nuclear export signal (NES) and...

The lysosomal enzyme cathepsin-D (cath-D) and insulin-like growth factor-II (IGF-II), which share a common IGF-II/mannose-6-phosphate (M6P) transmembrane receptor, are both synthesized and secreted by breast cancer cells, upon which they might exert an intracrine/autocrine control on proliferation. We have evaluated the binding of 125I-immunopurified human cath-D in different breast cell membrane preparations. The concentration of high affinity M6P reversible binding sites (mean Kd, 0.85 nM) varied among the different breast cancer cells (0-0.82 pmol/mg membrane protein), but there was no correlation between the presence of steroid receptor and M6P-dependent binding. Cross-linking experiments with [125I]cath-D and [125I]IGF-II showed the formation of complexes with the 270,000 mol wt IGF-II/M6P receptor molecule which migrated, respectively, at 330,000 and 270,000 mol wt in 3-10% gradient sodium dodecyl sulfate-polyacrylamide gels. [125I]IGF-II cross-linking was increased by M6P (20% above control), whereas cath-D strongly inhibited IGF-II interaction by 80%. Conversely, IGF-II reduced [125I]cath-D cross-linking by 55%. Direct ligand binding on receptors transferred onto nitrocellulose sheets by Western blotting confirmed the interaction of both ligands on the same receptor molecule. By studying IGF-II's growth-promoting activity in these cells in a wide range of concentrations, we show that IGF-II triggers its mitogenic response via IGF-II/M6P receptor at low concentrations, whereas it is mainly acting via IGF-I receptor at high concentrations. Three lines of evidences lead us to that conclusion.(ABSTRACT TRUNCATED AT 250 WORDS)

SummaryInteractions 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  chain of LRP1, and is the first ligand of the extracellular domain of LRP1 to be identified. We show that pro-cath-D interacts with LRP1 in cellulo. Interaction occurs at the cell surface, and overexpressed LRP1 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-LRP1 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.

Studies in Jurkat leukemia cells have suggested that protein-tyrosine phosphatase PTPL1/FAP-1 rescues Fasinduced cell death. However, we have previously shown that this enzyme triggers 4-hydroxytamoxifen-induced growth inhibition in human breast cancer cells. The present study addresses the role of PTPL1/FAP-1 in antiestrogen-regulated apoptotic effect and insulin-like growth factor-I survival action in MCF7 cells and further identifies the impacted signaling pathway. By terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling and cytoplasmic nucleosome enzyme-linked immunosorbent assay, we demonstrated that 4-hydroxytamoxifen-induced apoptosis was totally lost in PTPL1/FAP-1 antisense transfectants in which enzyme expression was abrogated, revealing the crucial role of this phosphatase in the apoptotic process in human breast cancer cells. Time-dependent expression of PTPL1/FAP-1 in MCF7 cells completely abolished the survival action of insulin-like growth factor-I. This effect occurred through a highly significant reduction in phosphatidylinositol 3-kinase/Akt pathway activation (80% reduction in phosphatidylinositol 3-kinase activity, 55% inhibition of Akt activation) accompanied by a 65% decrease in insulin receptor substrate-1 growth factor-induced tyrosine phosphorylation. These results provide the first evidence that PTPL1/FAP-1 has a key role in the apoptotic process in human breast cancer cells independent of Fas but associated with an early inhibition of the insulin receptor substrate-1/phosphatidylinositol 3-kinase pathway. Our data therefore suggest new therapeutic routes and strengthen the importance of identifying endogenous regulators and substrates of this phosphatase in breast tumors.Breast cancer is one of the most common malignancies affecting women in the Western countries. Although mortality and survival rates decreased in the UK (1), breast cancer incidence is still increasing. It is therefore crucial in the coming years to design new therapeutic strategies based on the updated knowledge of the mechanisms by which tumor growth is sustained.Breast cancer proliferation is the result of the balance between cell division and cell apoptosis. It has been shown in vitro in human breast cancer cell models that steroid hormones (mostly estrogens) and growth factors (epidermal growth factor, transforming growth factor ␣, IGF-I 1 and II, etc.) are the major signals affecting proliferation (2, 3). Most of these factors stimulate cell division and/or promote cell survival by mechanisms yet poorly understood, thus conferring growth advantage to tumor-responsive cells. On the other side, growth inhibitors and various antagonists have been shown to increase breast cancer cell apoptosis (4, 5).Estrogens and their antagonists mediate their action through their nuclear receptors (estrogen receptors ␣ and ␤), acting as transcriptional factors in coordination with numerous additional transcriptional cofactors. Peptide hormones and growth factor-signaling pathways involve the activation of sev...