Cathepsin-D is an independent marker of poor prognosis in human breast cancer. We previously showed that human wild-type cathepsin-D, as well as its mutated form devoid of proteolytic activity stably transfected in 3Y1-Ad12 cancer cells, stimulated tumor growth. To investigate the mechanisms by which human cathepsin-D and its catalytically-inactive counterpart promoted tumor growth in vivo, we quantified the expression of proliferating cell nuclear antigen, the number of blood vessels and of apoptotic cells in 3Y1-Ad12 tumor xenografts. We first verified that both human wild-type and mutated cathepsin-D were expressed at a high level in cathepsin-D xenografts, whereas no human cathepsin-D was detected in control xenografts. Our immunohistochemical studies then revealed that both wild-type cathepsin-D and catalytically-inactive cathepsin-D, increased proliferating cell nuclear antigen expression and tumor angiogenesis. Interestingly, wild-type cathepsin-D significantly inhibited tumor apoptosis, whereas catalytically-inactive cathepsin-D did not. We therefore propose that human cathepsin-D stimulates tumor growth by acting -directly or indirectly -as a mitogenic factor on both cancer and endothelial cells independently of its catalytic activity. Our overall results provide the first mechanistic evidences on the essential role of cathepsin-D at multiple tumor progression steps, affecting cell proliferation, angiogenesis and apoptosis.

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.

Cathepsin-D, a lysosomal aspartyl proteinase, is highly secreted by breast cancer cells and its over-expression by transfection stimulates cancer cell proliferation. The mechanism by which this protease a ects proliferation remains, however, unknown. In order to determine whether proteolytic activity is necessary, we abolished its enzymatic activity using site-directed mutagenesis followed by stable transfection in 3Y1-Ad12 cancer cells. Substitution of the aspartic acid residue 231 by an asparagine residue in its catalytic site abrogated the cathepsin-D proteolytic activity but did not a ect its expression level, processing or secretion. However, like wild-type cathepsin-D, this mutated catalytically-inactive cathepsin-D retained its capacity to stimulate proliferation of cells embedded in Matrigel or collagen I matrices, colony formation in soft agar and tumor growth in athymic nude mice. Addition on the mocktransfected cells, of either conditioned media containing the wild-type or the mutated pro-cathepsin-D, or of the puri®ed mutated pro-cathepsin-D, partially mimicked the mitogenic activity of the transfected cathepsin-D, indicating a role of the secreted pro-enzyme. Moreover, addition of two anti-cathepsin-D antibodies on the cathepsin-D transfected cells inhibited their proliferation, suggesting an action of the secreted pro-cathepsin-D via an autocrine loop. A synthetic peptide containing the 27-44 residue moiety of the cathepsin-D pro-fragment was, however, not mitogenic suggesting that a receptor for the pro-fragment was not involved. Furthermore, the cathepsin-D mitogenicity was not blocked by inhibiting the interaction of pro-cathepsin-D with the mannose-6-phosphate receptors. Our results altogether demonstrate that a mutated cathepsin-D devoid of catalytic activity is still mitogenic and suggest that it is acting extracellularly by triggering directly or indirectly a yet unidenti®ed cell surface receptor. Oncogene (2001) 20, 6920 ± 6929.

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.

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Estrogen receptor (ER)-positive breast cancers generally have a better prognosis and are often responsive to anti-estrogen therapy, which is the first example of a successful therapy targeted on a specific protein, the ER. Unfortunately ER-negative breast cancers are more aggressive and unresponsive to anti-estrogens. Other targeted therapies are thus urgently needed, based on breast cancer oncogene inhibition or suppressor gene activation as far as molecular studies have demonstrated the alteration of expression, or structure of these genes in human breast cancer. Using the MDA-MB.231 human breast cancer cell line as a model of ER-negative breast cancers, we are investigating two of these approaches in our laboratory. Our first approach was to transfect the ER or various ER-deleted variants into an ER-negative cell line in an attempt to recover anti-estrogen responsiveness. The unliganded receptor, and surprisingly estradiol, were both found to inhibit tumor growth and invasiveness in vitro and in vivo. The mechanisms of these inhibitions in ER-negative cancer cells are being studied, in an attempt to target the ER sequence responsible for such inhibition in these cancer cells. Another strategy is trying to inhibit the activity or expression of an oncogene specifically overexpressed in most breast cancers. This approach was recently shown by others to be efficient in breast cancer therapy with HER2-Neu oncogene amplification using Herceptin. Without excluding other molecular putative targets, we have focused our research on cathepsin D as a potential target, since it is often overexpressed in aggressive human breast cancers, including ER-negative tumors, and rarely associated with HER2-Neu amplification. Our first results obtained in vitro on cell lines and in vivo in tumor xenografts in nude mice, illustrate that the mode of action of cathepsin D in breast cancer is useful to guide the development of these therapies. In the past 20 years we have learned that the action of cathepsin D is complex and involves both intracellular and extracellular activities due to its proteolytic activity and to interactions with membrane components without catalytic activity. Each of these mechanisms could be potentially inhibited in an attempt to prevent tumor growth. Breast cancer is a very heterogeneous and multigenic disease and different targeted therapies adapted to each category of breast cancer are therefore required. Validated assays in the primary tumor of molecular markers such as ER, HER2-Neu and cathepsin D should help to predict which targeted therapy should be applied to cure breast cancer patients.

Defects in insulin secretion, resulting from loss of function or destruction of pancreatic ␤-cells, trigger diabetes. Interleukin (IL)-1␤ is a proinflammatory cytokine that is involved in type 1 and type 2 diabetes development and impairs ␤-cell survival and function. Because effective insulin signaling is required for the optimal ␤-cell function, we assessed the effect of IL-1␤ on the insulin pathway in a rat pancreatic ␤-cell line. We show that IL-1␤ decreases insulin-induced tyrosine phosphorylation of the insulin receptor (IR) and insulin receptor substrate (IRS) proteins as well as phosphatidylinositol 3-kinase (PI3K) activation, and that this action is not due to the IL-1␤-dependent nitric oxide (NO) production in RINm5F cells. We next analyzed if suppressor of cytokine signaling (SOCS)-3, which can be induced by multiple cytokines and which we identified as an insulin action inhibitor, was implicated in the IL-1␤ inhibitory effect on insulin signaling in these cells. We show that IL-1␤ increases SOCS-3 expression and induces SOCS-3/IR complex formation in RINm5F cells. Moreover, we find that ectopically expressed SOCS-3 associates with the IR and reduces insulin-dependent IR autophosphorylation and IRS/PI3K pathway in a way comparable to IL-1␤ treatment in RINm5F cells. We propose that IL-1␤ decreases insulin action in ␤-cells through the induction of SOCS-3 expression, and that this effect potentially alters insulin-induced ␤-cell survival. Diabetes 53 (Suppl. 3)

1 High concentrations of 5-hydroxytryptamine (5-HT) were shown to produce a positive chronotropic effect in the rat isolated atria associated with an increase in [3H] release when the atria were preloaded with [3H]-noradrenaline ([3H]-NA). The present study investigates the mechanism of this [3H] release using different antagonists.2 In atria preloaded with ['HI-NA (37 kBq ml-l, 0.4 pM, 20 min), 5-HT induced a rise in [3H] release values which appeared at the concentration of 2.5 pM and increased slightly with concentration ( 5 , 10, 20 and 50 p~) .However, 5-HT (2.5 pM) had no chronotropic effect while the different concentrations of 5, 10,20 and 50 pM induced the same increase in atrial rate (AR). By contrast, when MA0 activity was blocked by pargyline, 5-HT (2.5 pM) induced an increase in AR.3 In the absence of 5-HT, the 5-HT2 receptor antagonist, ketanserin, largely increased the spontaneous [3H] release without modifying the AR. The AR was increased by ketanserin in the presence of pargyline. The NA uptake inhibitor, desipramine, increased both [3H] release and AR. On the other hand, the incubation of atria with the 5-HT, and 5-HT2 receptor antagonist, methiothepin, the 5-HT3 receptor antagonist, zacopride or with one of the two specific 5-HT uptake inhibitors, fluvoxamine or indalpine, did not modify neither the [3H] efflux nor the AR. 4 In the presence of 5-HT (50 pM), methiothepin decreased the 5-HT-induced [3H] release and had no effect on the increase in AR due to 5-HT. By contrast, ketanserin reduced the [3H] increase induced by 5-HT. Zacopride did not modify the 5-HT-induced increases in [3H] release and in AR, while desipramine abolished completely these effects. Fluvoxamine and indalpine had no effect on the 5-HT chronotropic action. However, indalpine but not fluvoxamine decreased the 5-HT-induced [3H] release.5 It is concluded that a tyramine-like effect is the main mechanism of the 5-HT-induced chronotropic action at high concentrations. This effect is not modulated by the 5-HT3 receptors but involves the 5-HT, and/or the 5-HT2 receptor activity. The high concentrations of 5-HT protect the NA released from inactivation by MAO.