Cathepsin D is partly endocytosed by the LRP1 receptor and inhibits LRP1-regulated intramembrane proteolysis.

The aspartic protease cathepsin-D (cath-D) is a marker of poor prognosis in breast cancer that is overexpressed and hypersecreted by human breast cancer cells. Secreted pro-cath-D binds to the extracellular domain of the ß-chain of the LDL receptor-related protein-1 (LRP1) in fibroblasts. The LRP1 receptor has an 85-kDa transmembrane ß-chain and a noncovalently attached 515-kDa extracellular α-chain. LRP1 acts by (1) internalizing many ligands via its α-chain, (2) activating signaling pathways by phosphorylating the LRP1ß-chain tyrosine and (3) modulating gene transcription by regulated intramembrane proteolysis (RIP) of its ß-chain. LRP1 RIP involves two cleavages: the first liberates the LRP1 ectodomain to give a membrane-associated form, LRP1ß-CTF, and the second generates the LRP1ß-intracellular domain, LRP1ß-ICD, that modulates gene transcription. Here, we investigated the endocytosis of pro-cath-D by LRP1 and the effect of pro-cath-D/LRP1ß interaction on LRP1ß tyrosine phosphorylation and/or LRP1ß RIP. Our results indicate that pro-cath-D was partially endocytosed by LRP1 in fibroblasts. However, pro-cath-D and ectopic cath-D did not stimulate phosphorylation of the LRP1ß-chain tyrosine. Interestingly, ectopic cath-D and its catalytically inactive (D231N)cath-D, and pro-(D231N)cath-D all significantly inhibited LRP1 RIP by preventing LRP1ß-CTF production. Thus, cath-D inhibits LRP1 RIP independently of its catalytic activity by blocking the first cleavage. As cath-D triggers fibroblast outgrowth by LRP1, we propose that cath-D modulates the growth of fibroblasts by inhibiting LRP1 RIP in the breast tumor microenvironment.

Overexpression of both catalytically active and -inactive cathepsin D by cancer cells enhances apoptosis-dependent chemo-sensitivity.

The aspartic protease cathepsin D (cath-D) is a key mediator of induced-apoptosis and its proteolytic activity has been generally involved in this event. During apoptosis, cath-D is translocated to the cytosol. Because cath-D is one of the lysosomal enzymes that requires a more acidic pH to be proteolytically active relative to the cysteine lysosomal enzymes such as cath-B and -L, it is therefore open to question whether cytosolic cath-D might be able to cleave substrate(s) implicated in the apoptotic cascade. Here, we have investigated the role of wild-type cath-D and its proteolytically inactive counterpart overexpressed by 3Y1-Ad12 cancer cells during chemotherapeutic-induced cytotoxicity and apoptosis, as well as the relevance of cath-D catalytic function. We demonstrate that wild-type or mutated catalytically inactive cath-D strongly enhances chemo-sensitivity and apoptotic response to etoposide. Both wild-type and mutated inactive cath-D are translocated to the cytosol, increasing the release of cytochrome c, the activation of caspases-9 and -3 and the induction of a caspase-dependent apoptosis. In addition, pretreatment of cells with the aspartic protease inhibitor, pepstatin A, does not prevent apoptosis. Interestingly therefore, the stimulatory effect of cath-D on cell death is independent of its catalytic activity. Overall, our results imply that cytosolic cath-D stimulates apoptotic pathways by interacting with a member of the apoptotic machinery rather than by cleaving specific substrate(s).

A mutated cathepsin-D devoid of its catalytic activity stimulates the growth of cancer cells.

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 affects 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 affect 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 mock-transfected cells, of either conditioned media containing the wild-type or the mutated pro-cathepsin-D, or of the purified 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 extra-cellularly by triggering directly or indirectly a yet unidentified cell surface receptor.

Serum induction of the fibroblast growth factor-binding protein (FGF-BP) is mediated through ERK and p38 MAP kinase activation and C/EBP-regulated transcription.

The fibroblast growth factor-binding protein (FGF-BP) modulates FGF activity through binding and release from the extracellular matrix. Consequently, the expression of FGF-BP in certain tumor types is a rate-limiting regulator of FGF-mediated angiogenesis. FGF-BP is upregulated in squamous cell carcinoma by treatment with mitogens such as EGF or TPA. In this study, we investigated the regulation of FGF-BP gene expression by serum. Treatment of serum-starved ME-180 cells with fetal bovine serum (FBS) resulted in a rapid increase in steady-state levels of FGF-BP mRNA and in the rate of FGF-BP gene transcription. Serum induction of FGF-BP mRNA was not mediated through EGF receptor activation but was dependent on PKC, as well as ERK kinase (MEK) and p38 MAP kinase activation. Promoter analysis showed that C/EBP is the main promoter element required for the serum response. Unlike EGF-activation of FGF-BP, transcriptional induction by serum is not significantly regulated through the AP-1 or E-box sites in the promoter. These results illustrate differences between the mechanism of induction in response to serum and EGF.

Differential regulation of a fibroblast growth factor-binding protein by receptor-selective analogs of retinoic acid.

We have demonstrated earlier that a secreted fibroblast growth factor-binding protein (FGF-BP) can enhance angiogenesis and promote tumor growth in vivo. Furthermore, we found that FGF-BP expression in squamous cell carcinoma (SCC) is reduced by concentrations of retinoids that are effective in the treatment of SCC and that this repression can occur at the transcriptional and post-transcriptional level. To further examine the mechanism of regulation of FGF-BP by retinoids and the role played by retinoid receptor subtypes, we utilized retinoic acid receptor (RAR)-selective (TTNPB) and retinoid X receptor (RXR)-selective (LG100268) ligands. In ME-180 SCC cells, FGF-BP mRNA was down-regulated by TTNPB with an IC(50) value of 1 nM, whereas transcription was only repressed at 10,000-fold higher concentrations (IC(50) > 10 microM). This suggests that the major effects of retinoids on FGF-BP occur at the post-transcriptional level. In four additional SCC cell lines, FGF-BP was also down-regulated by TTNPB with IC(50) values of

Cathepsin D in cancer metastasis: a protease and a ligand.

Cathepsin D (cath-D) overexpression in breast cancer cells is associated with increased risk of metastasis in patients according to several clinical studies. No alterations of pro-cath-D structure or activation have been demonstrated in cancer cells. However, overexpression and dysrouting of pro-cath-D in illegitimate compartments could have consequences on tumor progression. Transfection of a human cDNA cath-D expression vector increases the metastatic potential of 3Y1-Ad12 embryonic rat tumorigenic cells when intravenously injected into nude mice. The mechanism by which cath-D increases the incidence of clinical metastasis seems to involve increased cell growth and decreased contact inhibition rather than escape of cancer cells through the basement membrane. Different mechanisms are discussed by which cath-D could act as a protease following its activation or as a ligand of different membrane receptors at a more neutral pH.

Phorbol ester-induced transcription of a fibroblast growth factor-binding protein is modulated by a complex interplay of positive and negative regulatory promoter elements.

Earlier studies from our laboratory showed that a secreted binding protein for fibroblast growth factors (FGF-BP) is expressed at high levels in squamous cell carcinoma (SCC) cell lines. Overexpression studies or conversely reduced expression of FGF-BP by ribozyme targeting have elucidated a direct role of this protein in angiogenesis during tumor development. We have also observed a significant up-regulation of FGF-BP during TPA (12-O-tetradecanoylphorbol-13-acetate) promotion of skin cancer. Here we investigate the mechanism of TPA induction of FGF-BP gene expression in the human ME-180 SCC cell line. We found that TPA increased FGF-BP mRNA levels in a time- and dose-dependent manner mediated via the protein kinase C signal transduction pathway. Results from actinomycin D and cycloheximide experiments as well as nuclear transcription assays revealed that TPA up-regulated the steady-state levels of FGF-BP mRNA by increasing its rate of gene transcription independently of de novo protein synthesis. We isolated the human FGF-BP promoter and determined by deletion analysis that TPA regulatory elements were all contained in the first 118 base pairs upstream of the transcription start site. Further mutational analysis revealed that full TPA induction required interplay between several regulatory elements with homology to Ets, AP-1, and CAATT/enhancer binding protein C/EBP sites. In addition, deletion or mutation of a 10-base pair region juxtaposed to the AP-1 site dramatically increased TPA induced FGF-BP gene expression. This region represses the extent of the FGF-BP promoter response to TPA and contained sequences recognized by the family of E box helix-loop-helix transcription factors. Gel shift analysis showed specific and TPA-inducible protein binding to the Ets, AP-1, and C/EBP sites. Furthermore, distinct, specific, and TPA-inducible binding to the imperfect E box repressor element was also apparent. Overall, our data indicate that TPA effects on FGF-BP gene transcription are tightly controlled by a complex interplay of positive elements and a novel negative regulatory element.

A secreted FGF-binding protein can serve as the angiogenic switch in human cancer.

The growth and metastatic spread of cancer is directly related to tumor angiogenesis, and the driving factors need to be understood to exploit this process therapeutically. However, tumor cells and their normal stroma express a multitude of candidate angiogenic factors, and very few specific inhibitors have been generated to assess which of these gene products are only innocent bystanders and which contribute significantly to tumor angiogenesis and metastasis. Here we investigated whether the expression in tumors of a secreted fibroblast growth factor (FGF)-binding protein (FGF-BP) that mobilizes and activates locally stored FGFs (ref. 11) can serve as an angiogenic switch molecule. Developmental expression of the retinoid-regulated FGF-BP gene is prominent in the skin and intestine during the perinatal phase and is down-modulated in the adult. The gene is, however, upregulated in carcinogen-induced skin tumors, in squamous cell carcinoma (SCC) and in some colon cancer cell lines and tumor samples. To assess the significance of FGF-BP expression in tumors, we depleted human SCC (ME-180) and colon carcinoma (LS174T) cell lines of their endogenous FGF-BP by targeting with specific ribozymes. We found that the reduction of FGF-BP reduced the release of biologically active basic FGF (bFGF) from cells in culture. Furthermore, the growth and angiogenesis of xenograft tumors in mice was decreased in parallel with the reduction of FGF-BP. This suggests that human tumors can utilize FGF-BP as an angiogenic switch molecule.

In vivo inhibition of angiogenesis and induction of apoptosis by retinoic acid in squamous cell carcinoma.

Retinoids inhibit the growth and reverse aberrant differentiation of squamous cell carcinoma (SCC) cells in vitro. To investigate the potential mechanisms of antitumor activity of retinoids in vivo, we used the cervical SCC cell line ME-180 as a s.c. tumor xenograft in athymic nude mice. After s.c. injection, tumor cells were allowed to form visible tumors and antitumor activity of all-trans-retinoic acid (tRA) was studied. tRA was administered daily for a 1-week or a 2-week period at 60 mg/kg/day. Tumor specimens were then analyzed using immunohistochemical staining for the number of blood vessels and apoptotic cells and for proliferating cell nuclear antigen expression. Furthermore, we studied the effect of the tRA treatment on the expression of a binding protein for fibroblast growth factors (BP; Gen-Bank accession no. M60047) that is a candidate angiogenesis modulator in SCC (F. Czubayko et al., J. Biol. Chem., 269: 28243-28248, 1994). We found that in vivo tRA treatment reduces BP expression in SCC xenografts, inhibits their angiogenesis, induces apoptosis of the tumor cells, and leads to a decrease of the tumor growth rate. We speculate that the tRA down-regulation of BP is responsible for the reduction of angiogenesis.

A tetracycline-responsive promoter system reveals the role of a secreted binding protein for FGFs during the early phase of tumor growth.

We showed previously that a secreted binding protein for FGFs (BP) can induce tumor growth and angiogenesis of a non-tumorigenic human cell line (SW-13). To study the contribution of BP to different phases of tumor growth, we employed a regulated promoter system which is highly active in SW-13 cells and can be downregulated >20-fold by treatment with tetracycline. We demonstrate that expression of BP in SW-13 cells (SW-13/tetBP cells) induces colony formation in soft agar and tumor growth in athymic nude mice. Tetracycline downregulated BP expression in these cells and prevented their colony formation in soft agar. Continuous tetracycline treatment of animals suppressed BP expression in tumors grown from SW-13/tetBP cells and reduced growth of the xenografts. Initiation of tetracycline treatment after xenograft tumors had been established had no effect on further tumor expansion in spite of downregulated BP levels. These data suggest that BP expression plays a role mainly in the early stages of tumor progression.

Regulation of gene expression of a binding protein for fibroblast growth factors by retinoic acid.

Retinoids are potent regulators of growth and differentiation and have shown promise as chemotherapeutic agents against selected cancers in particular squamous cell carcinoma (SCC). Earlier studies from our laboratory showed that a secreted binding protein for fibroblast growth factors (BP) is expressed at high levels in SCC cell lines and tissue samples. Here we investigate whether retinoids affect BP gene expression in SCC. In six different human SCC cell lines, we found that all-trans-retinoic acid (tRA) down-regulated BP mRNA by 39-89% within 24 h. From this group of cell lines, we selected the ME-180 cell line for more detailed studies of the mechanisms of this regulation. tRA down-regulated BP mRNA in a time- and dose-dependent manner. The effect of tRA was reversible, and BP mRNA returned to control levels within 24 h after removal of tRA. We also measured BP mRNA half-life and performed nuclear run-on experiments to study if tRA down-regulates BP by destabilizing the mRNA and/or by decreasing the rate of transcription. BP mRNA in ME-180 cells is very stable with a half-life of >16 h, and tRA decreased BP mRNA with a half-time of 5 h. Actinomycin D and cycloheximide blocked the tRA effect, suggesting that transcriptional regulation as well as de novo protein synthesis contribute to this post-transcriptional regulation of BP mRNA levels. In addition, tRA decreased the rate of BP gene transcription by 2- to 3-fold within 1 h. We conclude that retinoids down-regulate BP gene expression by post-transcriptional as well as by transcriptional mechanisms.