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Biological and Clinical Significance of Cathepsin D in Breast Cancer Metastasis

Université de Montpellier I and Unité Hormones et Cancer, Montpellier, France

Key Words. Cathepsin D • Breast cancer • Metastasis • Prognosis • Phagocytosis • Growth inhibitors

Dr. Henri Rochefort, Université de Montpellier I and Unité Hormones et Cancer (U 148) INSERM, 60, rue de Navacelles, 34090 Montpellier, France.

	Abstract

Top Abstract Introduction Structure and Normal Function... Alterations of Cath-D Expression... Cath-D as a Prognostic... Experimental Cath-D... Site of Pro-Cath-D Activation:... Search for Specific Cath-D... Conclusions Acknowledgements References

 
Cathepsin D (cath-D) is an aspartyl lysosomal protease expressed in all tissues. Most metastatic breast cancer cell lines, unlike normal cells, secrete high levels of pro-cath-D. This abnormal secretion is due to both overexpression of the cath-D gene and to an altered processing of the precursor protein. Cath-D gene transcription is increased by estrogen and growth factors in estrogen-receptor-positive breast cancer cells and by an unknown mechanism in estrogen-receptor-negative cells. A large number of independent clinical studies associated high cath-D concentrations in the cytosol of primary breast cancers with increased risk of subsequent metastasis. The amino acid sequence of cath-D analyzed in two breast cancer cell lines is normal, but glycosylation appears to be different with more acidic isoforms. To assess the potential role of this protease in cancer metastasis, we transfected a human cDNA cath-D expression vector in 3Y1-Ad12 embryonic rat tumorigenic cells which did not secrete the proenzyme. A moderate overexpression of human cath-D was sufficient to increase the metastatic potential of these cells in nude mice. The mechanism of cath-D-induced metastasis seems to require maturation of the proenzyme, in endosomes and in large acidic compartments identified as phagosomes. Rather than increase cancer cell escape from the primary tumor through basement membrane degradation as proposed for neutral porteinases, cath-D appears to facilitate cell growth at distant sites. The mechanism of this indirect mitogenic effect is discussed from results obtained in different models. Different cath-D substrates (growth inhibitors, precursors of growth factors, etc.) are proposed to mediate this activity.

	Introduction

Top Abstract Introduction Structure and Normal Function... Alterations of Cath-D Expression... Cath-D as a Prognostic... Experimental Cath-D... Site of Pro-Cath-D Activation:... Search for Specific Cath-D... Conclusions Acknowledgements References

 
Cath-D (E.C. 3.4.23.5), an aspartyl endoproteinase, has long been thought to be ubiquitously distributed in lysosomes of all cells in order to degrade proteins at an acidic pH in synergy with other cathepsins [1]. In addition to this general proteolytic action in lysosomes, cath-D may have a crucial role in activating proteins in prelysosomal compartments. For instance, it has been proposed that cath-D might play a role in antigen processing [2, 3], cell proliferation and tissue renewal [4] and activation of different prohormones [5-7].

Recently, double knock-out of this gene has shown that the homozygous mouse embryo developed normally. When pups were weaned, they began to lose weight and died postnatally at day 26 [4]. Two major alterations have been observed in the small intestine (necrosis and hemorrhage) and thymus (increased apoptosis). In contrast, the half-life of bulk proteins was not changed. This indicates that cath-D is a limiting factor for normal remodeling and renewal of some tissues but not for overall protein degradation in lysosomes. It has therefore been suggested that cath-D is required to provide essential growth factors for renewal of certain epithelial tissues. During prenatal development of cath-D deficient mice, cath-D could either be replaced by other proteases, or might be provided by the mother, at first from the plasma and later the milk.

Moreover, the role of cath-D in various pathological processes such as degenerative brain diseases [8], connective tissue disease [9] and cancer progression is now intensively investigated. Studies in estrogen-receptor-positive breast cancer cell lines have revealed that cath-D is both a housekeeping enzyme and a regulated enzyme induced by estrogens and growth factors. Its overexpression in some solid tumors is correlated with the metastatic potential of these tumors [10]. In the multistep process leading to metastasis, it has generally been proposed that proteases are mainly involved in the degradation of the basement membrane surrounding the primary tumor. This concept is largely documented for neutral proteases, such as plasminogen activators and collagenases which are activated extracellularly at the plasma membrane level. The case of cathepsins may not be as simple, since their action would require an acidic pH. Moreover, most recent results show that even though extracellular matrix (ECM) is potential substrate for cath-D, the major action may be via substrates leading to increased cell proliferation of tumor cells at distant sites.

	Structure and Normal Function of Cath-D

Top Abstract Introduction Structure and Normal Function... Alterations of Cath-D Expression... Cath-D as a Prognostic... Experimental Cath-D... Site of Pro-Cath-D Activation:... Search for Specific Cath-D... Conclusions Acknowledgements References

 
In humans, the cath-D gene containing nine exons is located in chromosome 11p15 and expresses a single transcript of 2.2 kb [11, 12]. Cath-D, like other aspartyl proteases such as renin, chymosin, and pepsinogen, has a bilobed structure [13]. Cath-D differs from these other proteases by its cellular routing to lysosomes. After addition of mannose-6 phosphate (M6P) by an N-acetylglucosaminyl phosphotransferase which recognizes specific sequences of the glycosylated proenzyme, a second enzyme, N-acetylglucoaminylphosphate glycosidase uncovers the blocked M6P residues [14, 15]. Kornfield et al. have shown that several parts of the proenzyme are required for its recognition by the phosphotransferase [15]. A cath-D precursor bearing M6P signals is sorted in the trans Golgi network by specific M6P receptors (MPRs).

Two distinct MPRs have been identified. The first, M6P/IGF-II receptor, is a large transmembrane glycoprotein of 300 kDa that also specifically binds insulin-like growth factor II but not IGF-I. The second, a cation-dependent receptor of 46 KDa, displays an extracytoplasmic domain homologous to each of the 15 repeats forming the extracellular domain of the M6P/IGF-II receptor [14, 15]. The respective roles of both receptors have been clarified when cells, lacking either receptor type, were generated by gene disruption experiments in mice. Cath-D is preferentially secreted by fibroblasts lacking the M6P/IGF-II receptor, indicating that this receptor is more important to target cath-D to lysosomes than the 46 kDa receptor [16]. Moreover, the M6P/IGF-II receptor can mediate endocytosis of secreted lysosomal enzymes and IGF-II, whereas the small receptor is inefficient in endocytosis. The large receptor is thought to be involved in degrading the excess of IGF-II in lysosomes.

	Alterations of Cath-D Expression and Targeting in Human Breast Cancer

Top Abstract Introduction Structure and Normal Function... Alterations of Cath-D Expression... Cath-D as a Prognostic... Experimental Cath-D... Site of Pro-Cath-D Activation:... Search for Specific Cath-D... Conclusions Acknowledgements References

 
Overexpression and Regulation of Cath-D Gene by Estrogen and Growth Factors
The major cath-D characteristic in breast cancer is its overexpression, which has been described both at the mRNA and protein levels [17-19]. Several approaches, such as immunohistochemistry, in situ hybridization, cytosolic immunoassay and Northern and Western blot analyses have indicated that in most breast cancer tumors cath-D is overexpressed 2- to 50-fold compared to its concentration in other cells such as fibroblasts or normal mammary glands [17]. Overexpressed cath-D areas are mostly located in breast cancer tissue and not in tumor fibroblasts, as shown by immunohistochemistry [20] and RNA in situ hybridization [21]. Although macrophages present in the surrounding tumor tissue also produce this enzyme, they do not seem to explain such a high cath-D concentration [20]. Two series of questions that arise from this overexpression concern its mechanism and consequences. In contrasts with several oncogenes such as c-erB2, the mechanism does not seem to involve gene amplification or major rearrangements that have not been detected in the few tumors analyzed to date [12].

In ER-positive breast cancer, both estrogen and growth factors stimulate cath-D protein and mRNA accumulation [18, 19]. As for other steroid-responsive genes, the regulation of cath-D mRNA accumulation by estrogen is mostly due to increased initiation of transcription [19, 22], but will not be detailed here. Estrogen-responsive elements have been defined in the proximal promoter region of the gene. In synergy with other regulatory sequences (SP1, AP1, etc.) they may be responsible for the stimulation of cath-D gene expression [23]. Since estrogen and growth factors stimulate the growth of ER-positive tumors, the induction of this protease appears to be associated with this growth stimulation. Other estrogen-induced proteins such as pS2 [24], the progesterone receptor [25] and eventually the wild type BrCa 1 protein [26] are associated with a good prognostic outcome. Contrary to these genes, cath-D is also overexpressed in ER-negative breast cancer [18]. The association of ER-negative breast cancer with high cath-D expression may be of particularly bad prognostic significance.

Structure of Breast Cancer Cath-D
To define the causes of its higher secretion in cancer cells, several studies have compared the protein structure, glycosylation and proteolytic activity of cath-D in normal and cancer cells. The nucleotide sequence of pro-cath-D has been determined in only two breast cancer cells lines (MCF7 and ZR75.1). It was found to be almost identical to the cDNA sequence of human kidney cath-D [12, 27]. In MCF7 cells, only one nucleotide modification at position 224 changes alanine to valine in propeptide [12]. A study of this change in several breast cancer and normal tissues of the same patients indicates that it was not consecutive to a somatic event, but to a polymorphism [28].

The glycosylation of the tumoral cath-D from MCF7 cells and of cath-D from normal tissues has been compared. In both cases, cath-D bears two N-linked oligosaccharide chains with M6P signals at their extremities. However, secreted tumoral pro-cath-D and its cellular processed forms are totally sensitive to endo-ß-N-acetyl glucosaminidase H (Endo H), whereas cellular cath-D from normal tissues is partially Endo H-resistant [29]. Furthermore, the secreted proenzyme is markedly heterogeneous and has a more acidic pH in MCF7 cells than in normal mammary cells. These acidic forms disappear following Endo H treatment, indicating that the structural difference between pro-cath-D of normal and cancer mammary cells is located on high mannose or hybrid N-oligosaccharides [17]. However, the nature of acidic components and the common occurrence of these differences in other breast cancers, as well as their biological importance, are currently unknown.

Proteolytic activity of cath-D from breast cancer cells appears to be similar if not identical to that of normal cells. Acidic pH is required from its activity in vitro with an optimum pH at 4.5-5.0 when tested on an extracellular matrix as substrate. The proenzyme can be auto-activated by partial or total removal of the profragment when present in an acidic microenvironment [30, 31].

Altered Targeting and Secretion
Another consequence of cath-D overexpression is the increased secretion of its proenzyme. In several breast cancer cell lines, pro-cath-D sorting is altered in comparison with normal mammary cells. This alteration results in lower cytoplasmic accumulation of the mature enzyme (34 kDa) and increased secretion of the proenzyme in cancer cells [17]. In some antiestrogen-resistant breast cancer cell lines, the secretion level of newly-synthesized pro-cath-D can reach 90% [32, 33].

This altered targeting does not seem to be due to a lack of M6P signals or a specific decreased affinity of pro-cath-D for an M6P receptor. In estrogen-responsive cells, while pro-cath-D and IGF-II are markedly increased by estrogens, the M6P/IGF-II receptor is downregulated by the hormone. We have thus proposed that an M6P receptor-saturation mechanism is responsible for hypersecretion of pro-cath-D (and other lysosomal proenzymes) following estrogen treatment [34]. However, the M6P/IGF-II receptor is not the only cath-D carrier to lysosomes. In addition to the 46 kDa M6P receptor, an M6P-independent membrane-bound routing has been described in macrophages [35] and HepG2 [36] cells, and may also be important in breast cancer cells, as recently observed in experiments involving NH₄C1 treatment [37]. By increasing the pH in sorting endosomes, this weak base prevents dissociation of the M6P/IGF-II receptor-cath-D complex and subsequent M6P receptor recycling [35]. In normal fibroblasts and mammary cells, the newly synthesized pro-cath-D normally sorted by the M6P/IGF-II receptor is secreted after NH₄C1 treatment but not accumulated in the cell. In many breast and ovarian cancer cells, the proenzyme level after NH₄C1 treatment increases in the cell but not in the conditioned medium. This resistance to NH₄C1 appears to be pro-cath-D specific, since in the same cell line other enzymes such as ß-hexosaminidase, -glucosidase and arylsufatase are not resistant [37].

	Cath-D as a Prognostic Marker in Breast Cancer

Top Abstract Introduction Structure and Normal Function... Alterations of Cath-D Expression... Cath-D as a Prognostic... Experimental Cath-D... Site of Pro-Cath-D Activation:... Search for Specific Cath-D... Conclusions Acknowledgements References

 
Cath-D in breast cancer is overexpressed by 2- to 50-fold compared to its concentration in other cells, such as fibroblasts or normal mammary glands [17]. Both the mRNA and the protein levels are increased, as shown by immunohistochemistry, in situ hybridization, cytosolic immunoassay and Northern and Western blot analyses. Overexpressed cath-D areas are located in breast cancer tissue and not in tumor fibroblasts, as shown by immunohistochemistry [20] and RNA in situ hybridization [21]. The mechanism of this overexpression does not seem to involve gene amplification or major chromosomal rearrangements [12]. Clearly, most of the clinical studies from different and independent clinical centers using a standardized and validated cytosolic immunoassay, based on two monoclonal antibodies, indicate that the cath-D level in primary breast cancer cytosol is an independent prognostic parameter associated with occurrence of clinical metastases and shorter survival [38].

Quantification of the cath-D proteolytic activity in the cytosol after activation at an acidic pH gave similar results [39]. In contrast, studies performed by not-yet standardized in situ immunohistochemistry assays or Western blot analysis are mostly discordant. These discrepancies could be due to the use of different tissue fixations and antibodies, absence of quantification or loss of the secreted form of cath-D during fixation [40]. Immunohistochemical assay of cath-D is also complicated by the fact that the relative contribution of infiltrating macrophages and cancer cells is not clear. We found that the cytosolic level is more correlated with cancer cell staining than the number of macrophages [20], but other groups using different antibodies point to the importance of production by stromal cells [41].

Another reservation concerns node negative breast cancer when the prognostic cath-D value has not always been found, contrary to its value in the general population. It might be important, however, to measure this protease to complement the axillary lymph node status whose reliability depends on the skill and experience of the pathologist, and which always results in nonaesthetic scars and sometimes sequelae to the patient.

Moreover, in most studies, this prognostic parameter, sometimes associated with lymph node status, is independent of the other parameters, including the estrogen receptor (ER) Urokinase or its inhibitor, as shown in a recent study of 2,500 patients from Rotterdam [42]. Like other strong prognostic markers, its determination will be indispensable as soon as its significance in terms of response to adjuvant therapy is specified.

Whether high cath-D level is also associated with bad prognosis in other solid tumors remains to be elucidated even though its overexpression has been described in other cancers such as melanoma [43], hepatoma [44], colorectal [45] and prostatic [46] carcinoma.

From our earlier clinical studies showing an unexpected correlation between high cath-D concentration and metastasis [47], we tried to determine whether the increased cath-D concentration found in primary cancer could be a cause or was only an associated characteristic of tumors able to develop clinical metastases.

	Experimental Cath-D Overexpression Increases Colonization and Distant Metastasis through its Proteolytic Activity

Top Abstract Introduction Structure and Normal Function... Alterations of Cath-D Expression... Cath-D as a Prognostic... Experimental Cath-D... Site of Pro-Cath-D Activation:... Search for Specific Cath-D... Conclusions Acknowledgements References

 
The potential role of cath-D in cancer metastasis was studied after stable transfection of an expression vector of human cath-D into a rat tumorigenic cell line (3Y1-Ad12) which does not secrete cath-D. The metastatic potential of cath-D-expressing clones was compared to that of control clones in athymic mice [48, 49]. Four cath-D clones isolated from two independent transfections displayed a higher metastatic potential than four corresponding controls. The incidence and size of gross liver metastases were significantly increased in mice injected with cath-D clones, while these clones expressed moderate human cath-D concentrations (two-fold lower than the median concentration detected in human primary breast cancers). This was the first direct evidence that cath-D overexpression accelerates the appearance of clinically detectable metastases.

The mechanism of cath-D action on metastasis could involve either its interaction with the plasma membrane M6P/IGF-II receptor or its catalytic activity. To assess this question, the endoplasmic reticulum retention signal, KDEL, was inserted by mutagenesis at the C-terminal of the cath-D coding sequence [49]. The consequence of pro-cath-D retention in the endoplasmic reticulum was to prevent its maturation in vitro and in vivo and to abolish its effects on metastases. We verified that a control peptide. KDAS, inserted at the same position prevented neither the maturation nor the stimulation of the metastasis potency. This inactivation of cath-D by modification of two amino acids (KDEL versus KDAS) strongly supports a direct and specific action of cath-D on the metastatic process and excludes artifacts due to transfection or selection procedure. Moreover, we also show that the metastatic potential was unaffected in some KDEL clones secreting high concentrations of pro-cath-D-KDEL which displayed the same affinity to the M6P/IGF-II receptor as the wild-type cath-D. We conclude that pro-cath-D interaction with the M6P/IGF-II receptor at the plasma membrane is not sufficient to stimulate metastasis and that maturation of cath-D into an active protease is a major requisite for its action on metastasis. Therefore, cath-D appears to act as a protease and not as a ligand transducing signal via a membrane receptor.

	Site of Pro-Cath-D Activation: Intracellular, Extracellular or Both?

Top Abstract Introduction Structure and Normal Function... Alterations of Cath-D Expression... Cath-D as a Prognostic... Experimental Cath-D... Site of Pro-Cath-D Activation:... Search for Specific Cath-D... Conclusions Acknowledgements References

 
We have established that maturation of the enzyme is required in the rat tumor cell model. Maturation also occurs in vivo in primary breast cancers since separate assays of pro-cath-D and total cath-D using different monoclonal antibodies showed that the tumor contained only 4% to 6% of pro-cath-D, while the metastatic breast cancer cell lines secrete up to 50% of the precursor in vitro [50]. One major question is where maturation occurs in the human tumor.

Intracellular Activation of Pro-Cath-D and Phagocytosis
As opposed to other tissue proteinases, no cath-D tissue inhibitor is known. The major requirement for pro-cath-D to be autoactivated seems to be its localization in an acidic microenvironment [30, 31]. We have therefore searched for such acidic compartments, different from lysosomes, in which cath-D could partially digest or liberate molecules playing a decisive role in cancer cell growth and/or invasion. Such as acidic milieu has been previously described in pre-lysosomal compartments such as late endosomes [35, 36]. There are several examples in the literature showing that cath-D activates specific substrates in endosomes. The activated substrates, either a prohormone or a transmembrane receptor, could later exert their transmembrane-signaling function in endosomes [51].

Searching for extracellular acidic compartments, we actually found large intracellular acidic vesicles (LAVs) of 5 µm in diameter. These LAVs are more frequently found in vitro in breast cancer cells than in normal mammary cells and contain high levels of cath-D but no pro-cath-D [52]. LAVs were not restricted to cell lines, since they were also found in primary cultures of pleural effusions from breast cancer and in paraffin sections of cancer biopsies [20]. Using MDA-MB231 breast cancer cells, we found more LAV-positive cells after migration through Matrigel than before migration [53]. These vesicles contained phagocytosed extracellular material such as latex beads or pieces of extracellular matrix, and can therefore be considered as large heterophagosomes. The fact that breast cancer cells are fully able to phagocytose extracellular material, including extracellular matrix, and to digest this material within heterophagosomes is unusual, since this activity is known to occur mainly in specialized phagocytotic cells such as macrophages and polynuclear neutrophils. The phagocytotic activity of cancer cells has been previously considered [54] and supported by in vitro invasion of chicken blastoderm by cancer cells engulfing hypoblast yolk [55]. Heterophagocytosis associated with high proton secretion [53] may facilitate development of cancer cell colonies in distal parenchyma. In phagosomes, however, the large amount of engulfed extracellular material needs to be actively digested by cathepsins [38]. This could explain why cancer cells overexpressing cath-D have a higher capability in developing metastasis.

Extracellular Activation of Pro-Cath-D at the Contact of Basement Membrane
Most proteases are believed to play a role in degrading basement membrane following secretion and extracellular activation [56]. Cancer cells can then cross the basement membrane border to invade the stroma and extravasate in blood. This mechanism had been initially thought to be unlikely since very acidic pH (at least 5.5) is required to activate pro-cath-D, and this acidic pH is generally found in intracellular compartments. However, one major characteristic of cath-D and other cathepsins (B, L, etc.) in cancer cells is their increased secretion. The mechanism of this secretion is not yet clear. It is unlikely to be due to an alteration of the structure of the protease but could be due to a specific alteration of the routing mechanism of pro-cathepsin in general, involving either the Man-6-P/IGF-II receptor [34] or an alternative routing [35-37] or both. After secretion, pro-cath-D could potentially be activated extracellularly. Extracellular pH in tumors is generally more acidic than that in the corresponding normal tissue. We recently found that breast cancer cells as macrophages have a high potential for liberating protons in the extracellular milieu through lactic acid production and a functional H+/ATPase pump at the plasma membrane level. They are able to decrease the pH to 5.5 when it is measured under MCF7 cell monolayers and when the acidifying potential of the invasive MDA-MB231 breast cancer cells is more important than in the estrogen-responsive MCF7 cells (Montcourrier P., Silver I.A., Rochefort H., submitted for publication).

	Search for Specific Cath-D Substrates Involved in Cancer Metastasis

Top Abstract Introduction Structure and Normal Function... Alterations of Cath-D Expression... Cath-D as a Prognostic... Experimental Cath-D... Site of Pro-Cath-D Activation:... Search for Specific Cath-D... Conclusions Acknowledgements References

 
Cath-D in these acidic vesicles is able to intracellularly digest many types of proteins, including proteins of the engulfed ECM, and thus provide food, amino acid supply and space for invasive breast cancer cells. Cath-D also has the potential to degrade or activate many important molecules able to play a role in one of the steps leading to metastasis.

It has been proposed that cath-D can trigger the proteolytic cascade leading to ECM degradation by activating pro-cathepsin B and/or degrading cystatins [57]. However, most of our studies point to a role of cath-D in facilitating distant metastasis via an indirect mitogenic activity rather than stimulation of local invasion and extravasation [48, 49]. The mitogenic activity of cath-D initially shown in MCF7 cells cultured in monolayer using purified pro-cath-D [58] is debated in vitro [59] but confirmed in vivo following cDNA transfection [48, 60]. The inactivation of cath-D gene by homologous recombination (knock-out mice) shows that cath-D is not required for normal prenatal development, but the 25-day old mice die rapidly after weaning [4]. The lack of cath-D induces necrosis in epithelial small intestine and apoptosis in thymocytes. This strongly supports the idea that cath-D facilitates epithelial cell growth during tissue remodeling. Also, the original approach of Ann Chamber's group based on in vivo videomicroscopic observation of fluorescent cancer cells indicated that the limiting step for metastasis was not intravasation of cancer cells, their survival in the circulation or their extravasation, but the ability of cancer cells to form colonies at distant sites in a foreign environment [61]. When transported to distant sites, cancer cells should find and utilize foods and produce the enzyme(s) allowing them to multiply and form colonies in these sites. Cath-D facilitates the production of nutrients such as amino acids from the ECM but could also favor cancer cell proliferation via specific inactivation of growth inhibitors and activation (or liberation) of growth factors.

Inactivation of Secreted Growth Inhibitors(s)
It has been proposed that IGF-Binding Protein 3 can be specifically destroyed by cath-D, thus liberating IGF-I to stimulate its receptor and the growth of cancer cells [62]. We showed recently in our rat tumor model system transfected with human cath-D-cDNA that cath-D could also inactivate growth inhibitors. Stable cath-D transfected clones in low-serum conditions reached 2- to 4.5-fold higher density at confluence than control clones [48]. Control cells reaching saturation density release an inhibitory activity that is able to prevent growth of control or cath-D clones. In contrast, this growth inhibitory activity was markedly reduced in cath-D clones [60]. Therefore, cath-D overexpression increases cell density by inhibiting the activity or secretion of growth inhibitors released by confluent cells. Cath-D probably acts intracellularly since the addition of the secreted proenzyme had no effect on the saturation density and no mature cath-D was detectable in the culture medium. Moreover, maturation of the intracellular proenzyme seems necessary since neutralization of acidic compartments by chloroquine or ammonium chloride prevented both cath-D maturation and its mitogenic effect. Further studies are in progress to identify the secreted growth inhibitor(s).

Liberation of Growth Factors from ECM
Several growth factors and cytokines are entrapped in an inactive form in the ECM, including pro-TGFß, which may facilitate tumor cell invasion, and factors of the fibroblast growth factor (FGF) family which display a high angiogenic activity.

We previously showed that MCF7 breast cancer cells, cultured on an ECM secreted by bovine endothelial cells, were able to digest this matrix and liberate biologically active ¹²⁵I bFGF which had been preincorporated into the matrix. This bFGF could be incorporated into MCF7 cells, but not in the presence of pepstatin A, indicating that in more biological culture conditions, an aspartyl protease, most likely cath-D, was activated to degrade ECM and liberate bFGF [63]. Consequently, the liberated bFGF could both stimulate the growth of cancer cells and increase angiogenesis by stimulating the growth of surrounding endothelial cells.

	Conclusions

Top Abstract Introduction Structure and Normal Function... Alterations of Cath-D Expression... Cath-D as a Prognostic... Experimental Cath-D... Site of Pro-Cath-D Activation:... Search for Specific Cath-D... Conclusions Acknowledgements References

 
Cath-D overexpression in some cancer cells is not only correlated with, but also facilitates, the development of distant metastases from circulating cancer cells. This has been demonstrated in rat tumor cell systems by transfecting human cath-D gene, and may also occur in humans on the basis of the clinical value of cath-D level as a prognostic parameter of breast cancer.

The experimental systems used to study cath-D mechanisms have varied tremendously from simple in vitro systems, either cell-free with one enzyme and one substrate, or with only one type of cell, to more complex systems, taking into account paracrine regulations and cancer cell interactions with ECM.

Using different experimental approaches, our laboratory found that cath-D activity is able, mostly intracellularly, to stimulate cancer cell growth at high density by inactivating a secreted growth inhibitor [60], and/or by liberating growth and angiogenic factors from the ECM [63] and/or by providing amino acid following phagocytosis of ECM [52, 53]. These mechanisms are not mutually exclusive but seem to result more in an increased growth of distant micrometastasis than in a stimulation of local invasion by digestion of basement membrane. However, the respective involvement of these mechanisms in vivo remains to be demonstrated. The data in cath-D deficient mice [4] indicating that cath-D is required for renewal of certain tissues agrees with the concept that cath-D overexpression facilitates epithelial cancer cell growth at distant sites. Further work aimed at inhibiting cath-D action in vivo on these targets is necessary to find new therapeutic approaches for preventing or inhibiting the occurrence of metastases.

	Acknowledgements

Top Abstract Introduction Structure and Normal Function... Alterations of Cath-D Expression... Cath-D as a Prognostic... Experimental Cath-D... Site of Pro-Cath-D Activation:... Search for Specific Cath-D... Conclusions Acknowledgements References

 
This work was supported by the "Institut National de la Santé, et de la Recherche Médicale," the University of Montpellier I, the "Ligue Nationale Contre le Cancer," the "Association pour la Recherche sur le Cancer," and the "Conseil Régional du Languedoc-Roussillon."

	References

Top Abstract Introduction Structure and Normal Function... Alterations of Cath-D Expression... Cath-D as a Prognostic... Experimental Cath-D... Site of Pro-Cath-D Activation:... Search for Specific Cath-D... Conclusions Acknowledgements References

 

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