Estrogen-induced lysosomal proteases secreted by breast cancer cells: A role in carcinogenesis?

In an attempt to understand the mechanism by which estrogens stimulate cell proliferation and mammary carcinogenesis, metastatic human breast cancer cell lines (MCF7, ZR75-1) were found to secrete a 52,000 dalton (52K) protein under estrogen stimulation. Following its purification to homogeneity, the 52K protein was identified as a secreted procathepsin-D-like aspartyl protease bearing mannose-6-phosphate signals. This precursor displays an in vitro autocrine mitogenic activity on estrogen-deprived MCF7 cells and is able to degrade basement membrane and proteoglycans following its autoactivation. The total protease (52K + 48K and 34K) was detected and assayed by monoclonal antibodies and was found to be highly concentrated in proliferative and cystic mastopathies. In breast cancer, its cytosolic concentration appears to be correlated more to tumor invasiveness than to hormone responsiveness. The mRNA of the 52K protease accumulates rapidly following estradiol treatment, as was shown by Northern blot analysis with cloned cDNA. The 52K cathepsin-D-like protease is the first example of a lysosomal protease induced by estrogens in cancer cells. Results obtained using different approaches suggest that two cysteinyl cathepsins are also related to cell transformation and invasiveness. It has been proposed that cathepsin-B is involved in breast cancer and metastatic melanoma, and its regulation by estrogen has been shown in the rat uterus. Cathepsin-L corresponds to the major excreted protein (MEP) whose synthesis and secretion are markedly increased by transformation of NIH 3T3 cells with Ki ras and are regulated by several growth factors. In addition to secreted autocrine growth factors and to other proteases (plasminogen activator, collagenase), lysosomal cathepsins may therefore play an important role in the process of tumor growth and invasion as long as their precursor is secreted abundantly.     

The estrogen-regulated 52K-cathepsin-D in breast cancer: From biology to clinical applications

We have studied estrogen-regulated proteins in an attempt to understand the mechanism by which estrogens stimulate cell proliferation and mammary carcinogenesis. In estrogen receptor positive human breast cancer celllines (MCF7, ZR75-1) estrogens specifically increase the production into the culture medium of a 52,000 daltons (52K) glycoprotein. Several high affinity monoclonal antibodies to the partially purified secretory 52K protein have allowed to purify to homogeneity this protein and its cellular processed products. The 52K protein has been identified as the secreted precursor of a cathepsin-D like protease bearing mannose-6-phosphate signals and routed to lysosomes via mannose-6-phosphate receptor. The protease is mitogenic in vitro on estrogen deprived MCF7 cells and is able to degrade basement membrane and proteoglycans following its activation. The cellular related proteins, as detected by immunohistochemistry and immunoassay are more concentrated in proliferative mammary ducts than in resting ducts and their concentration in breast cancer cytosol appears to be more correlated with lymph nodes invasion and disease free survival (with S. Thorpe, Copenhagen) than with the estrogen receptor (RE) level. The protein is also produced constitutively by RE-negative cell lines, while in some antiestrogen resistant variants, it becomes inducible by tamoxifen, contrary to the wild type MCF7 cells. Cloning of its cDNA in λgt11 has allowed to show that the mRNA is rapidly induced by estrogens and to sequence the protein and compare it to that of the normal human kidney cathepsin-D. We conclude that the estrogen-induced cathepsin-D like protease may have important autocrine and/or paracrine functions in stimulating the growth and invasion of hormone-dependent and independent breast cancers and may be useful as a tissue marker to predict high risk mastopathies and breast cancer invasiveness, In addition to other estrogen regulated growth factors, the precursor of lysosomal proteases has the potential to stimulate the proliferation and invasiveness of breast cancer cells as long as they are secreted in excess rather than being routed to lysosomes.     

Purification and first characterization of the secreted and cellular 52-kDa proteins regulated by estrogens in human-breast cancer cells

An estrogen-regulated 52-kDa glycoprotein secreted by MCF7 breast cancer cells was first purified from serum-free conditioned medium by concanavalin-A--Sepharose (ConA--Sepharose). The 13% pure protein was then used to obtain monoclonal antibodies to the 52-kDa protein [Garcia et al. (1985) Cancer Res. 45, 709-716]. Using ConA--Sepharose and monoclonal antibody affinity chromatographies, the secreted 52-kDa protein was finally purified to homogeneity as verified by silver staining of sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) and one single N-terminal amino acid. The purification factor was approximately 1400 and the yield 40%. The same two-step procedure, applied to MCF7 cell extracts, yielded four immunologically related proteins of 52 kDa, 48 kDa, 34 kDa and 17 kDa, which were purified 1250-fold with a yield of 30%. These components were further separated by high-performance liquid chromatography gel filtration under denaturing conditions. The final products were homogeneous on the basis of silver-stained SDS-PAGE and gel filtration. However, isoelectrofocusing showed that the pI of the secreted 52-kDa protein and the cellular 34-kDa protein varied from 5.5 to 6.5. Amino acid analysis of the secreted and the related cellular 34-kDa protein is given. Western immunoblotting, pulse chase studies and post-translational studies indicate that the 52-kDa protein is the precursors of a lysosomal enzyme which is partially secreted and partially processed into smaller cellular forms.     

The 52-kDa estrogen-induced protein secreted by MCF7 cells is a lysosomal acidic protease

An estrogen-induced 52-kDa glycoprotein secreted by human breast cancer cells and able to autostimulate the growth of MCF7 cells has been purified, using monoclonal antibodies, and characterized. The protein contains mannose 6-phosphate signals on its N-linked high-mannose chains, suggesting that it is a lysosomal enzyme. Both the secreted 52-kDa protein and its processed cellular forms (52-, 48- and 34-kDa) were identified as carboxyl proteinases having an optimal activity at pH 3.5 and being specifically inhibited by pepstatin. This protease is characterized by its inducibility by estrogens and its high concentration in proliferative benign and malignant mammary tissue, when detected by immunohistochemistry. The estrogen-induced secretion of this protease may help to understand how estrogens stimulate mammary tumor growth and/or invasion.     

Phosphorylation, glycosylation, and proteolytic activity of the 52-kD estrogen-induced protein secreted by MCF7 cells

We have studied the posttranslational modifications of the 52-kD protein, an estrogen-regulated autocrine mitogen secreted by several human breast cancer cells in culture (Westley, B., and H. Rochefort, 1980, Cell, 20:353-362). The secreted 52-kD protein was found to be phosphorylated mostly (94%) on high-mannose N-linked oligosaccharide chains, and mannose-6-phosphate signals were identified. The phosphate signal was totally removed by alkaline phosphatase hydrolysis. The secreted 52-kD protein was partly taken up by MCF7 cells via mannose-6-phosphate receptors and processed into 48- and 34-kD protein moieties as with lysosomal hydrolases. By electron microscopy, immunoperoxidase staining revealed most of the reactive proteins in lysosomes. After complete purification by immunoaffinity chromatography, we identified both the secreted 52-kD protein and its processed cellular forms as aspartic and acidic proteinases specifically inhibited by pepstatin. The 52-kD protease is secreted in breast cancer cells under its inactive proenzyme form, which can be autoactivated at acidic pH with a slight decrease of molecular mass. The enzyme of breast cancer cells, when compared with cathepsin D(s) of normal tissue, was found to be similar in molecular weight, enzymatic activities (inhibitors, substrates, specific activities), and immunoreactivity. However, the 52-kD protein and its cellular processed forms of breast cancer cells were totally sensitive to endo-beta-N-acetylglucosaminidase H (Endo H), whereas several cellular cathepsin D(s) of normal tissue were partially Endo H-resistant. This difference, in addition to others concerning tissue distribution, mitogenic activity and hormonal regulation, strongly suggests that the 52-kD cathepsin D-like enzyme of breast cancer cells is different from previously described cathepsin D(s). The 52-kD estrogen-induced lysosomal proteinase may have important functions in facilitating the mammary cancer cells to proliferate, migrate, and metastasize.     

Structure, function, regulation and clinical significance of the 52K pro-cathepsin D secreted by breast cancer cells

In estrogen-receptor-positive human breast cancer cell lines (MCF7, ZR75-1), estrogens specifically increase the secretion into the culture medium of a 52,000 Da (52K) glycoprotein and stimulate cell proliferation. The 52K protein has been purified to homogeneity using monoclonal antibodies and identified as the secreted precursor of a cathepsin D bearing mannose-6-phosphate signals. The secreted precursor 52K protein is mitogenic in vitro in estrogen-deprived MCF7 cells, can be taken up by these cells via mannose-6-phosphate receptors, and can degrade extracellular matrix and proteoglycans following its auto-activation. The protease is also produced constitutively by ER-negative cell lines, and is inducible by tamoxifen in some antiestrogen-resistant variants. The corresponding cDNA has been cloned using N-terminal sequencing of the protein and monoclonal antibodies. Its complete sequencing indicates a strong homology with pro-cathepsin D of normal tissues. Using a cDNA probe, the regulation of 52K cathepsin D mRNA by estrogens and antiestrogens has been studied and chromosome localization determined by in situ hybridization. Clinical studies using both immunohistochemistry and immunoenzymatic assay of breast cancer cytosol have shown that the concentration of total cellular cathepsin D (52K + 48K + 34K) is related to the proliferation of mammary ducts and to the prognosis of breast cancer. Its cytosolic concentration in primary tumors of postmenopausal patients is correlated slightly with lymph node invasion and significantly with shorter disease-free intervals in a 6-year retrospective study with the Danish Breast Cancer Groups and Finsen Institute (S. Thorpe et al.).(ABSTRACT TRUNCATED AT 250 WORDS)     

An Active 32-kDa Cathepsin L Is Secreted Directly from HT 1080 Fibrosarcoma Cells and Not via Lysosomal Exocytosis

Cathepsin L [EC 3.4.22.15] is secreted via lysosomal exocytosis by several types of cancer cells, including prostate and breast cancer cells. We previously reported that human cultured fibrosarcoma (HT 1080) cells secrete cathepsin L into the medium; this secreted cathepsin is 10-times more active than intracellular cathepsin. This increased activity was attributed to the presence of a 32-kDa cathepsin L in the medium. The aim of this study was to examine how this active 32-kDa cathepsin L is secreted into the medium. To this end, we compared the secreted active 32-kDa cathepsin L with lysosomal cathepsin L by using a novel gelatin zymography technique that employs leupeptin. We also examined the glycosylation and phosphorylation status of the proteins by using the enzymes endoglycosidase H [EC 3.2.1.96] and alkaline phosphatase [EC 3.1.3.1]. Strong active bands corresponding to the 32-kDa and 34-kDa cathepsin L forms were detected in the medium and lysosomes, respectively. The cell extract exhibited strong active bands for both forms. Moreover, both forms were adsorbed onto a concanavalin A-agarose column. The core protein domain of both forms had the same molecular mass of 30 kDa. The 32-kDa cathepsin L was phosphorylated, while the 34-kDa lysosomal form was dephosphorylated, perhaps because of the lysosomal marker enzyme, acid phosphatase. These results suggest that the active 32-kDa form does not enter the lysosomes. In conclusion, our results indicate that the active 32-kDa cathepsin L is secreted directly from the HT 1080 cells and not via lysosomal exocytosis.     

Procathepsin D interacts with prosaposin in cancer cells but its internalization is not mediated by LDL receptor-related protein

The cell surface binding, endocytosis, and lysosomal routing of procathepsin D (procath-D) in cancer cells are mostly independent of the mannose-6-phosphate (M6P) receptors. In an attempt to define the receptor involved, we intracellularly cross-linked procath-D with a 68-kDa protein that we identified with specific antibodies as prosaposin in human breast and ovarian cancer cell lines. In cancer cells, this protein-protein interaction was resistant to ammonium chloride or M6P treatment, indicating that it was independent of the M6P receptors. A similar interaction also occurred in the breast cancer cell culture medium between the secreted prosaposin and procath-D. Since these two precursors can be endocytosed, we then determined whether they were interacting with the same cell surface receptor. In fibroblasts, we confirmed that the endocytosis of these two proteins was different since it was generally mediated by the M6P receptors for procath-D and mostly by LRP (LDL receptor-related protein) for prosaposin. In breast cancer cells, prosaposin endocytosis was not detected, in contrast to procath-D endocytosis, suggesting that the majority of procath-D is not internalized as a complex with prosaposin. Moreover, RAP (receptor-associated protein), a ligand inhibiting LRP-mediated endocytosis, prevented internalization of prosaposin in 49-F rat fibroblasts, but did not affect procath-D M6P-independent internalization in MDA-MB231 cells. We conclude that in breast cancer cells, even though procath-D interacts intracellularly and extracellarly with prosaposin, it is endocytosed independent of prosaposin by a receptor different from the M6P receptors and the LRP.     

Regulation, clinical and biological significance of cathepsin D in breast cancer

The lysosomal protease, pro-cathepsin D, is overexpressed and secreted by human breast cancers. In estrogen-responsive breast cancer cell lines, estrogens and growth factors stimulate cathepsin D expression through distinct mechanisms. Clinical studies indicate that high cathepsin D concentration in primary breast cancers is correlated with an increased risk of metastasis and particularly useful to orientate node-negative tumors towards an adjuvant therapy.     

Purification and characterization of a novel growth factor from human breast cancer cells.

We have purified and characterized a novel 30-kDa glycoprotein (gp30) with TGF alpha-like properties secreted from the estrogen receptor negative breast cancer cell line MDA-MB-231. This factor was immunoprecipitated by an anti-TGF alpha polyclonal antibody and also had TGF alpha-like biological activity, as assayed by EGF radioreceptor assay and anchorage-independent assays. In addition, the novel growth factor stimulated phosphorylation of the EGF receptor and erbB-2 receptor. However, the novel growth factor, unlike EGF and TGF alpha, bound to heparin-Sepharose. Purification of gp30 was obtained to apparent homogeneity by heparin affinity chromatography and subsequent reversed-phase chromatography. Tunicamycin treatment in vivo or N-glycanase deglycosylation in vitro revealed a putative precursor of approximately 22 kDa molecular mass in contrast to the "normal" 16-kDa precursor species for TGF alpha. In vitro translation of total mRNA from MDA-MB-231 cells confirmed the size of the putative precursor. Biochemical characterization of gp30 was begun by V8 protease digestion of the deglycosylated polypeptide and the translated products. Peptide mapping of V8-digested, immunoprecipitated material suggests that the amino acid sequence of this unique protein is distinct from mature TGF alpha and not the result of a posttranslational modification of the precursor. We conclude that this TGF alpha-like (gp30) polypeptide is a novel growth factor with agonistic activity for both EGF and erbB-2 receptors.     

A 46-kDa antigen associated with estrogen receptor in human breast cancer

A 65-kDa estrogen receptor (ER) protein has been demonstrated both by sucrose gradient analysis and by immunoblot, using anti-ER monoclonal antibodies (MAbs). Since the ER is denatured in many experimental situations, such as formaldehyde fixing of samples for histochemistry and electroimmunoblotting studies, in this work we used a denatured 60-70-kDa ER-rich protein preparation as antigen for mice immunization in order to raise anti-ER MAbs. That material was obtained by affinity purification on an allyl-estradiol matrix of the MCF-7 cytosolic ER, followed by further isolation and enrichment by PAGE. NS-1 myeloma cells and spleen lymphocytes from the immunized mice were fused, and resultant hybridoma colonies were screened by [125I]-estradiol-labelled nuclear ER immunoprecipitation. The isolated MAb, E476, shows a moderate ability to precipitate ER and reacts strongly with a 46-kDa antigen in Western blot assay. The 46-kDa antigen was not detectable in native cytosol but became reactive after 50% ammonium sulfate precipitation of cytosolic proteins. The 46-kDa antigen appeared concentrated in the NaSCN plus estradiol eluate of the affinity column used for cytosolic ER purification. Freshly prepared 60-70-kDa material from the preparative gel electrophoresis did not show any E476 reactivity. However, when the 60-70-kDa proteins were frozen, thawed and speed vacuum concentrated, the 46-kDa antigen became detectable. Storage increased the reactivity of the 60-70-kDa material with the E476 MAb. The 46-kDa antigen was present only in the ER positive cell lines, and was absent in all negative cell lines tested. The 46-kDa protein is also present in the ER positive human breast cancer specimens. We conclude that the 46-kDa protein identified with the E476 MAb in human breast cancer is probably a naturally occurring ER fragment.     

A secreted glycoprotein induced by estrogen in human breast cancer cell lines

Estrogen induces the synthesis of a glycoprotein of molecular weight 46,000 daltons in three estrogen receptor-positive breast cancer cell lines (MCF₇, ZR₇₅ and T47D), but not in an estrogen receptor-negative cell line (BT 20) or a nonmalignant cell line (HBL 100). The 46K protein, which accounts for 40% of ³⁵S-methionine incorporation into secreted proteins, is only induced by steroids able to interact with the estrogen receptor. The anti-estrogens tamoxifen and hydroxytamoxifen, which by themselves were inactive, suppressed the induction of this protein by estradiol. In MCF₇ cells, estradiol also induces three intracellular proteins which are resolved in two-dimensional electrophoresis. The induction of the 46K secreted protein(s) makes these cell lines excellent in vitro systems for studying the mechanism of estrogen and anti-estrogen action. This protein may also be a useful probe for studying the action of estrogen on breast cancer growth, and may be a useful marker for predicting the hormonal responsiveness of breast cancer in vivo.     

A calcium-dependent protease associated with the neural cytoskeleton. Purification and partial characterisation

Calcium-dependent protease activity was found associated with a neurofilament-enriched cytoskeleton isolated from the bovine spinal cord. The protease was extracted from the cytoskeleton by 0.6 M KCl, and purified to apparent homogeneity (3300-fold) by chromatography on organomercurial-Sepharose 4B, casein-Sepharose 4B, and Sepharose CL-6B. A cytosolic calcium-dependent protease was similarly purified from the bovine spinal cord, after the cytosol was fractionated on DEAE-cellulose. Both cytoskeleton-bound and cytosolic enzymes had an apparent molecular mass of 100 kDa as judged by gel filtration, and consisted of two subunits (79 kDa and 20 kDa) upon sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Both enzymes exhibited caseinolytic activity with 0.5 mM Ca2+ and above, and the activity was strongly inhibited by various thiol protease inhibitors. In the presence of 0.1-0.2 mM Ca2+, the 68-kDa and 160-kDa components, and to a lesser extent the 200-kDa component, of the neurofilament triplet polypeptides were degraded by the cytosolic protease, whereas the cytoskeleton-bound protease needed two-fold higher concentration of Ca2+ to degrade the neurofilaments. Nevertheless, the cytoskeleton-bound protease in situ, i.e. before its extraction form the cytoskeleton by 0.6 M KCl, preferentially degraded the 160-kDa component in the presence of 0.1-0.2 mM Ca2+, suggesting that a proper locational relation of this enzyme to the neurofilament structure is a prerequisite to its preference for the 160-kDa component. It appears that a factor or factors involved in such an interaction between the protease and the neurofilament were eliminated during the course of enzyme purification. The glial fibrillary acidic protein was almost insensitive to the proteases purified in the present study.     

IGF-I affects glycosaminoglycan/proteoglycan synthesis in breast cancer cells through tyrosine kinase-dependent and -independent pathways

The insulin-like growth factor I (IGF-I) has been implicated in breast cancer development acting through insulin-like growth factor I receptor (IGF-IR), but also through estrogen receptor (ER). The effect of IGF on proteoglycan (PG) synthesis by two human breast cancer epithelial cell lines, the ER-positive MCF-7 and the ER-negative BT-20, was studied alone and in combination with genistein. Both cell lines synthesise hyaluronan (HA), matrix secreted and cell membrane-associated galactosaminoglycan containing proteoglycans (GalAGPGs) and heparan sulphate proteoglycans (HSPGs) in variable amounts. IGF-I affects the synthesis of PGs by BT-20 cells by decreasing the amounts of HA and secreted GalAGPGs and HSPGs and upregulates the expression of cell membrane-associated GalAGPGs and HSPGs. IGF-I exerts this effect on BT-20 cells acting mainly through receptors with protein tyrosine kinase activity (PTK). In contrast, IGF-I stimulates the synthesis of secreted GalAGPGs and HSPGs by MCF-7 cells, exhibiting only a slight suppression on synthesis of cell-associated GalAGPGs and HSPGs. The regulatory effect of IGF-I on PGs distribution in MCF-7 cells is mediated through a mix of pathways, which involves both receptors with PTK activity and PTK-independent signalling. It is suggested that the effects of IGF-I on the synthesis and distribution of PGs by epithelial breast cancer cells also depend on the presence or the absence of ER. The result of the IGF-I action is the balanced biosynthesis between the matrix and cell-associated PGs in both cell lines, approaching a common biosynthetic phenotype.     

The DEK Oncogene Is a Target of Steroid Hormone Receptor Signaling in Breast Cancer

Expression of estrogen and progesterone hormone receptors indicates a favorable prognosis due to the successful use of hormonal therapies such as tamoxifen and aromatase inhibitors. Unfortunately, 15–20% of patients will experience breast cancer recurrence despite continued use of tamoxifen. Drug resistance to hormonal therapies is of great clinical concern so it is imperative to identify novel molecular factors that contribute to tumorigenesis in hormone receptor positive cancers and/or mediate drug sensitivity. The hope is that targeted therapies, in combination with hormonal therapies, will improve survival and prevent recurrence. We have previously shown that the DEK oncogene, which is a chromatin remodeling protein, supports breast cancer cell proliferation, invasion and the maintenance of the breast cancer stem cell population. In this report, we demonstrate that DEK expression is associated with positive hormone receptor status in primary breast cancers and is up-regulated in vitro following exposure to the hormones estrogen, progesterone, and androgen. Chromatin immunoprecipitation experiments identify DEK as a novel estrogen receptor α (ERα) target gene whose expression promotes estrogen-induced proliferation. Finally, we report for the first time that DEK depletion enhances tamoxifen-induced cell death in ER+ breast cancer cell lines. Together, our data suggest that DEK promotes the pathogenesis of ER+ breast cancer and that the targeted inhibition of DEK may enhance the efficacy of conventional hormone therapies.     

IFN-gamma regulation of vacuolar pH, cathepsin D processing and autophagy in mammary epithelial cells

In this study we examined the ability of interferon-gamma (IFN-gamma) to regulate mammary epithelial cell growth and gene expression, with particular emphasis on two genes: Maspin (a member of serine protease inhibitor superfamily), and the lysosomal aspartyl endopeptidase cathepsin D (CatD). The protein products of these genes are critically involved in regulation of multitude of biological functions in different stages of mammary tissue development and remodeling. In addition, the expression of Maspin is down-regulated in primary breast cancer and is lost in metastatic disease, while CatD is excessively produced and aberrantly secreted by breast cancer cells. We report that IFN-gamma receptors are expressed in mammary epithelial cells, and receptor engagement by IFN-gamma transduces the IFN-gamma signal via Stat-1 resulting in decreased vacuolar pH. This change in vacuolar pH alters CatD protein processing and secretion concurrent with increased Maspin secretion. In addition, IFN-gamma exerts a suppressive effect on cell growth and proliferation, and induces morphological changes in mammary epithelial cells. Our studies also reveal that breast cancer cells, which are devoid of Maspin, are refractory to IFN-gamma with respect to changes in vacuolar pH and CatD. However, Maspin transfection of breast cancer cells partially sensitizes the cells to IFN-gamma's effect, thus providing new therapeutic implications.     

Cloning of cDNA sequences of a progestin-regulated mRNA from MCF7 human breast cancer cells.

A cDNA clone corresponding to an mRNA regulated by the progestin R5020, has been isolated by differential screening of a cDNA library from the MCF7 breast cancer cell line, which contains estrogen and progesterone receptors. This probe hybridized with a single species of poly A + RNA of 8-kb molecular weight as shown by Northern blot analysis and could also be used to total RNA preparation. This recombinant clone hybridized specifically to an mRNA coding for a 250,000 daltons protein when translated in vitro. This protein was identical to the 250 kDa progestin-regulated protein that we previously described (Biochem. Biophys. Res. Commun. 121, 421-427, 1984) as shown by immunoprecipitation with specific rabbit polyclonal antibodies. Dose-response curve and specificity studies show that the accumulation of the Pg8 mRNA and that of the 250-kDa protein was increased by 5 to 30-fold following progestin treatment and that this effect was mediated by the progesterone receptor. Time course of induction indicated that the accumulation of mRNA was rapid and preceded that of the protein. This is the first report on a cloned cDNA probe of progestin-regulated mRNA in human cell lines.