Phosphoproteomic analysis of Syk kinase signaling in human cancer cells reveals its role in cell-cell adhesion.

The spleen tyrosine kinase Syk has predominantly been studied in hematopoietic cells in which it is involved in immunoreceptor-mediated signaling. Recently, Syk expression was evidenced in numerous nonhematopoietic cells and shown to be involved in tumor formation and progression. The Syk downstream signaling effectors in nonhematopoietic cells remain, however, to be uncovered, and were investigated using MS-based quantitative phosphoproteomics. Two strategies, based on the inhibition of the Syk catalytic activity and on the loss of Syk expression were employed to identify phosphotyrosine-dependent complexes. Quantitative measurements were obtained on 350 proteins purified with phosphotyrosine affinity columns using the SILAC method. Forty-one proteins are dependent on both Syk expression and catalytic activity and were selected as signaling effectors. They are involved in a variety of biological processes such as signal transduction, cell-cell adhesion and cell polarization. We investigated the functional involvement of Syk in cell-cell adhesion and demonstrated the phosphorylation of E-cadherin and alpha-catenin. In addition, Syk is localized at cell-cell contacts, and Syk-mediated phosphorylation of E-cadherin seems to be important for the proper localization of p120-catenin at adherens junctions. Identification of the biochemical pathways regulated by Syk in human cancer cells will help to uncover its role in tumor formation and progression.

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.

Pathophysiology of limb girdle muscular dystrophy type 2A: hypothesis and new insights into the IkappaBalpha/NF-kappaB survival pathway in skeletal muscle.

Limb girdle muscular dystrophies (LGMDs) are a group of clinically heterogeneous genetic diseases characterized by progressive weakness and atrophy of scapular and pelvic muscles, with either a dominant or recessive autosomic mode of inheritance. The first symptoms of the disorder appear during the first 20 years of life and progresses gradually, and a walking disability develops 10-20 years later. The gene responsible for LGMD2A has been identified and encodes calpain 3, a protease expressed mainly in skeletal muscle. Apoptotic myonuclei were recently detected in muscular biopsy specimens of LGMD2A patients, and apoptosis was found to be correlated with altered subcellular distribution of inhibitory protein kappaBalpha (IkappaBalpha) and nuclear factor kappaB (NF-kappaB), resulting in sarcoplasmic sequestration of NF-kappaB. Calpain 3 dependent IkappaBalpha degradation was reconstituted in vitro, supporting a possible in vivo sequence of events leading from calpain 3 deficiency to IkappaBkappa accumulation, prevention of nuclear translocation of NF-kappaB, and ultimately apoptosis. Therefore calpain 3, present in healthy muscle as sarcoplasmic and nuclear forms, may control IkappaBalpha turnover and indirectly regulate NF-kappaB dependent expression of survival genes. Recent data reported from a new model of LGMD2A in mice and from other muscular disorders strengthen understanding of the molecular links between calpain 3 and the Ikappaalpha/NF-kappaB pathway. Finally, in light of the lack of apoptosis observed in inflammatory myopathies, a unifying model for the control of cell survival in muscle is proposed and discussed

In vitro invasion assays: phagocytosis of the extracellular matrix.

Degradation of the extracellular matrix is an essential component of phagocytosis by tumor cells and can be correlated with their invasive capacity. This unit presents flow cytometry based assays for rapid quantitative assessment of both proteolysis and internalization or internalization alone. These assays provide high-throughput, low-cost methods to compare cell lines and test the efficacy of treatments designed to stimulate or inhibit invasion.

Heregulin regulates the actin cytoskeleton and promotes invasive properties in breast cancer cell lines.

The metastatic process requires changes in tumor cell adhesion properties, cell motility and remodeling of the extracellular matrix. The erbB2 proto-oncogene is overexpressed in approximately 30% of breast cancers and is a major prognostic parameter when present in invasive disease. A ligand for the erbB2 receptor has not yet been identified but it can be activated by heterodimerization with heregulin (HRG)-stimulated erbB3 and erbB4 receptors. The HRGs are a family of polypeptide growth factors that have been shown to play a role in embryogenesis, tumor formation, growth and differentiation of breast cancer cells. The erbB3 and erbB4 receptors are involved in transregulation of erbB2 signaling. The work presented here suggests biological roles for HRG including regulation of the actin cytoskeleton and induction of motility and invasion in breast cancer cells. HRG-expressing breast cancer cell lines are characterized by low erbB receptor levels and a high invasive and metastatic index, while those which overexpress erbB2 demonstrate minimal invasive potential in vitro and are non-tumorigenic in vivo. Treatment of the highly tumorigenic and metastatic HRG-expressing breast cancer cell line MDA-MB-231 with an HRG-neutralizing antibody significantly inhibited proliferation in culture and motility in the Boyden chamber assay. Addition of exogenous HRG to non-invasive erbB2 overexpressing cells (SKBr-3) at low concentrations induced formation of pseudopodia, enhanced phagocytic activity and increased chemomigration and invasion in the Boyden chamber assay. The specificity of the chemomigration response to HRG is demonstrated by inhibition with the anti-HRG neutralizing antibody. These results suggest that either HRG can act as an autocrine or paracrine ligand to promote the invasive behavior of breast cancer cells in vitro or thus may enhance the metastatic process in vivo.

The Syk tyrosine kinase suppresses malignant growth of human breast cancer cells.

Syk is a protein tyrosine kinase that is widely expressed in haematopoietic cells. It is involved in coupling activated immunoreceptors to downstream signalling events that mediate diverse cellular responses including proliferation, differentiation and phagocytosis. Syk expression has been reported in cell lines of epithelial origin, but its function in these cells remains unknown. Here we show that Syk is commonly expressed in normal human breast tissue, benign breast lesions and low-tumorigenic breast cancer cell lines. Syk messenger RNA and protein, however, are low or undetectable in invasive breast carcinoma tissue and cell lines. Transfection of wild-type Syk into a Syk-negative breast cancer cell line markedly inhibited its tumour growth and metastasis formation in athymic mice. Conversely, overexpression of a kinase-deficient Syk in a Syk-positive breast cancer cell line significantly increased its tumour incidence and growth. Suppression of tumour growth by the reintroduction of Syk appeared to be the result of aberrant mitosis and cytokinesis. We propose that Syk is a potent modulator of epithelial cell growth and a potential tumour suppressor in human breast carcinomas.

Dual actions of sphingosine-1-phosphate: extracellular through the Gi-coupled receptor Edg-1 and intracellular to regulate proliferation and survival.

Sphingosine-1-phosphate (SPP), a bioactive lipid, acts both intracellularly and extracellularly to cause pleiotropic biological responses. Recently, we identified SPP as a ligand for the G protein-coupled receptor Edg-1 (Lee, M.-J., J.R. Van Brocklyn, S. Thangada, C.H. Liu, A.R. Hand, R. Menzeleev, S. Spiegel, and T. Hla. 1998. Science. 279:1552-1555). Edg-1 binds SPP with remarkable specificity as only sphinganine-1-phosphate displaced radiolabeled SPP, while other sphingolipids did not. Binding of SPP to Edg-1 resulted in inhibition of forskolin-stimulated cAMP accumulation, in a pertussis toxin-sensitive manner. In contrast, two well-characterized biological responses of SPP, mitogenesis and prevention of apoptosis, were clearly unrelated to binding to Edg-1 and correlated with intracellular uptake. SPP also stimulated signal transduction pathways, including calcium mobilization, activation of phospholipase D, and tyrosine phosphorylation of p125(FAK), independently of edg-1 expression. Moreover, DNA synthesis in Swiss 3T3 fibroblasts was significantly and specifically increased by microinjection of SPP. Finally, SPP suppresses apoptosis of HL-60 and pheochromocytoma PC12 cells, which do not have specific SPP binding or expression of Edg-1 mRNA. Conversely, sphinganine-1-phosphate, which binds to and signals via Edg-1, does not have any significant cytoprotective effect. Thus, SPP is a prototype for a novel class of lipid mediators that act both extracellularly as ligands for cell surface receptors and intracellularly as second messengers.

Phagocytosis of cross-linked gelatin matrix by human breast carcinoma cells correlates with their invasive capacity.

During invasion and metastasis, cancer cells interact closely with the extracellular matrix molecules by attachment, degradation, and migration. We demonstrated previously the local degradation of fluorescently labeled gelatin matrix by cancer cells at invasive membrane protrusions, called invadopodia. Using the newly developed quantitative fluorescence-activated cell sorting-phagocytosis assay and image analysis of localized degradation of fluorescently labeled matrix, we document here that degradation and site-specific removal of cross-linked gelatin matrix is correlated with the extent of phagocytosis in human breast cancer cells. A higher phagocytic capacity is generally associated with increasing invasiveness, documented in other invasion and motility assays as well. Gelatin phagocytosis is time and cell density dependent, and it is mediated by the actin cytoskeleton. Most of the intracellular gelatin is routed to actively acidified vesicles, as demonstrated by the fluorescent colocalization of gelatin with acidic vesicles, indicating the intracellular degradation of the phagocytosed matrix in lysosomes. We show here that normal intracellular routing is blocked after treatment with acidification inhibitors. In addition, the need for partial proteolytic degradation of the matrix prior to phagocytosis is demonstrated by the inhibition of gelatin phagocytosis with different serine and metalloproteinase inhibitors and its stimulation by conditioned medium containing the matrix metalloproteinases MMP-2 and MMP-9. Our results demonstrate that phagocytosis of extracellular matrix is an inherent feature of breast tumor cells that correlates with and may even directly contribute to their invasive capacity. This assay is useful for screening and evaluating potential anti-invasive agents because it is fast, reproducible, and versatile.

Integrin alpha 3 beta 1 participates in the phagocytosis of extracellular matrix molecules by human breast cancer cells.

The mechanisms and receptors involved in phagocytosis by nonhematopoietic cells are not well understood. The involvement of the alpha 3 beta 1 integrin in phagocytosis of the extracellular matrix by human breast cancer cells was studied. The possible role of this integrin was suggested since alpha 3 and beta 1 but not alpha 2 subunits are concentrated at membrane sites where local degradation of fluorescently labeled gelatin occurs. Strikingly, anti-alpha 3 integrin monoclonal antibodies (mAbs) stimulate the phagocytosis of fluorescently labeled gelatin films, gelatin beads, and Matrigel films in a quantitative phagocytosis assay. Stimulation of the gelatin uptake by the anti-alpha 3 mAb is dose responsive, saturable, and time dependent. Antibodies against other integrin subunits have a lower stimulatory effect (anti-beta 1) or no significant effect (anti-alpha 2, -alpha 5, -alpha 6, and -alpha v) on gelatin phagocytosis. The synthetic HGD-6 human laminin peptide that binds specifically the alpha 3 beta 1 integrin, but not the scrambled HSGD-6 control peptide, also markedly stimulates gelatin uptake in a dose-responsive way. Furthermore, the stimulatory effects of the HGD-6 peptide and the anti-alpha 3 mAb are additive, suggesting that they might promote phagocytosis in different ways. Other laminin (YIGSR, IKVAV) and fibronectin (GRGDS) peptides have no effect on gelatin phagocytosis. Immunofluorescence shows that the alpha 3 and the beta 1, but not the alpha 2 integrin subunit, concentrate into patches on the cell surface after treatment with their respective mAbs. And, both gelatin and the alpha 3 beta 1 but not the alpha 2 beta 1 integrin are cointernalized and routed to acidic vesicles such as lysosomes. In conclusion, we demonstrate that human breast cancer cells locally degrade and phagocytose the extracellular matrix and show for the first time that the alpha 3 beta 1 integrin participates in this phagocytosis. We hypothesize that the anti-alpha 3 antibodies and the laminin peptide HGD-6 activate the alpha 3 beta 1 integrin, which results in a downstream signaling cascade stimulating phagocytosis.

Anti-proliferative and anti-estrogenic effects of ICI 164,384 and ICI 182,780 in 4-OH-tamoxifen-resistant human breast-cancer cells.

The effects of the anti-estrogens 4-hydroxytamoxifen (OHTam), ICI 164,384 and ICI 182,780 were tested on the MCF-7/LCC2 breast-carcinoma cell line, which grows significantly in the presence of OHTam and serves as a model for studying anti-estrogen resistance of estrogen-receptor-positive breast cancer. Cell proliferation and cathepsin-D secretion were strongly inhibited by either ICI 182,780 or ICI 164,384 alone or ICI 164,384 in combination with 17-beta-estradiol (E2) or OHTam. ICI 164,384 alone did not affect the cathepsin-D and pS2 mRNA levels, but antagonized the stimulatory effects of E2 or OHTam on these 2 mRNAs. OHTam was more effective than E2 in increasing cathepsin-D mRNA levels, supporting the idea that anti-estrogen-resistant breast cancer continues to overexpress cathepsin-D. These data show that the steroidal anti-estrogens ICI 164,384 and ICI 182,780 retain their ability to inhibit cell proliferation and the estrogen-responsiveness of cathepsin-D and pS2 genes in the OHTam-resistant MCF-7/LCC2 cell lines. These pure anti-estrogens may thus be efficient second-line treatments of some Tamoxifen-resistant tumors.

Laminin binding and internalization by human and murine mammary gland cell lines in vitro.

We have studied the binding and internalization of Engelbreth-Holm-Swarm mouse sarcoma laminin labeled with colloidal gold (LN-G40) by human and murine mammary gland cell lines. Interactions between the LN-G40 probe and the cells spread on a glass coverslip were monitored with video-enhanced contrast microscopy (Nanovid). Transmission electron microscopy allowed the quantitation of the LN-G40 probe at various cellular locations. During the first 15 min, a homogeneous binding of LN-G40 probe to the cell surface was observed with all cell lines. This binding did not occur with gold particles that were not conjugated to laminin. Then, the LN-G40 probe began to cluster on the cell surface and was, during the following 20 h, internalized by pits that were not coated. In the cells, the LN-G40 probe sometimes showed saltatory movements along linear tracks. The LN-G40 probe was intracellularly found in vesicles, multivesicular bodies, cisternal structures, and lysosomes, suggesting the degradation of the internalized laminin. However, not all cell surface-bound LN-G40 probe was internalized after 20 h. Differences between the cell lines were quantitative, but no clear correlation could be made between migration of cells on laminin and internalization of laminin.

Arrest of MCF-7 cell migration by laminin in vitro: possible mechanisms.

Laminin, a major basement membrane component, arrested the migration of MCF-7/AZ human breast adenocarcinoma cells that were not invasive in vitro. Migration of invasive MCF-7/6 cells was not affected by laminin. Both cell types expressed the 67 kD laminin receptor, at both mRNA and protein level, but did not express the alpha 6 subunit of the VLA-6 integrin-type laminin receptor. The presence of YIGSR peptides (100 micrograms/ml), reported to block the interaction between laminin and its 67 kD receptor, did not change the migratory response of MCF-7/AZ or MCF-7/6 cells when meeting laminin lanes. In addition, the migration of these cell types was not affected by the presence of 17-beta-estradiol (10(-6) M) or all-trans retinoic acid (10(-6) M), which were both reported to increase the number of 67 kD receptors. We could therefore not assign an involvement of the 67 kD receptors in migration of MCF-7 cells on laminin, nor did we find evidence that conditioned medium of MCF-7/6 cells contains factors that are able to initiate migration of MCF-7/AZ cells on laminin.

Influence of basement membrane molecules on directional migration of human breast cell lines in vitro.

Spheroidal cell aggregates were prepared from four tumorigenic human breast cell lines (HBL-100 and three MCF-7 variants). Cells from these aggregates were allowed to migrate towards lanes of basement membrane components coated on a glass substratum. Matrigel (reconstituted basement membrane) lanes permanently arrested the migration of one MCF-7 cell line, while migration of the others was permitted. Amongst several purified basement membrane constituents only laminin, not collagen type IV or fibronectin, was found to cause the same arrest of migration. Within the laminin molecule only the pepsin P1, not the elastase E8 fragment, efficiently arrested migration of that cell line. Although migration was inhibited by these components, time-lapse video recordings revealed that arrested cells still proliferated and actively ruffled on top of the coatings. These data suggest that, amongst several basement membrane components, laminin can function as a stop signal for cell migration. Within laminin, this activity seems to be mainly associated with the P1 fragment. We conclude that laminin is the major determinant of the barrier-function of the basement membrane, to which some cell types have become insensitive.

Effect of oncogene transfection or passage in vivo on malignant phenotypes of rat2 cells.

Rat2 cells are thymidine kinase-deficient derivatives from the immortalized rat embryo cell line Rat1. They show no phenotypic correlates of malignancy in vitro and produce tumors in syngeneic Fischer rats after long latency periods. We have investigated how transfection with oncogenes would alter the in vitro and in vivo behavior of Rat2 cells. Thus we have manipulated Rat2 cultures in various ways. The cell lines obtained were categorized as parental, in vitro subclones, untransfected in vivo derivatives, non-oncogene (neor and tk) transfectants, oncogene (mutated c-Ha-ras, polyoma middle-T, FBR v-gag-fos-fox) transfectants, and in vivo derivatives of transfectants. They were tested in vitro for morphotype, colony formation in soft agar, growth in organ culture, invasion in organ culture, and in vivo for latency period of tumor formation, tumor growth rate, invasiveness, and metastasis. Differences between the consequences of various manipulations were found in the number of malignancy-related phenotypic alterations. The following trend could be deduced from our data: induction of invasiveness in organ culture by all manipulations; morphotypic transformation and shortening of tumor-latency period by all oncogene transfections and by passage with tumor formation in vivo; growth in organ culture and increased tumor growth rate in vivo by transfection with ras-, or fos-oncogenes and by passage in vivo. Metastatic capability (present in parental Rat2 cell tumors) and colony formation in soft agar (absent in Rat2 cells) were not affected by the present manipulations. We concluded that differences between the oncogene-transfectants and the untransfected in vivo derivatives do not lie in the expression of malignancy-related phenotypes but in the time needed to acquire them.

Tumorigenicity, invasiveness and metastatic capability of FR3T3 rat cells before and after transfection with bovine papilloma virus type 1 DNA.

Fischer rat FR3T3 cells were tested for tumorigenicity, invasive and metastatic capabilities before and after transfection, either with the entire bovine papilloma virus type 1 (BPV-1) genome or with a plasmid (pV69) containing a 69 per cent Bam H1-Hind III fragment of the BPV-1 genome as well as bacterial sequences. Cell lines were grouped as parental, pV69-transfectants, BPV-1 transfectants, in vitro derivatives, and in vivo derivatives. The tumorigenic, invasive and metastatic capabilities of these cell lines were examined in vivo through s.c., and i.p. injections of cell suspensions and through s.c. implantations of cellular aggregates into syngeneic rats. Invasiveness was tested in vitro through confrontations with embryonic chick heart fragments in organ culture. All cell lines including parental lines, were found to be invasive in vitro and tumorigenic in vivo; all tumors were invasive. It is, therefore, not possible to draw conclusions about the role of BPV-1 gene sequences in the acquisition of the invasive phenotype. Transfection with BPV-1 genes conveyed the metastatic phenotype upon parental FR3T3 cells, which were themselves found to be non-metastatic. With regards to this, no differences were found between BPV-1 transfectants compared with pV69 transfectants. Untransfected cells became metastatic also through passage in vivo as an s.c. tumor. The expression of the metastatic phenotype was not noticeably correlated with alterations of growth characteristics of the cell lines. We concluded that the implication of BPV-1 gene sequences in conveying the metastatic phenotype upon FR3T3, if any, was indirect, presumably through alterations of the host cell genome. Our experiments illustrate the need for long-term observations with parental cell lines before drawing conclusions about the role of oncogenes in the acquisition of the malignant phenotype.

The anti-invasive flavonoid (+)-catechin binds to laminin and abrogates the effect of laminin on cell morphology and adhesion.

To study the effect of the flavonoid (+)-catechin on cell-matrix interactions two cell types with a different morphology on and adhesion to laminin were used. MO4 virally transformed fetal mouse cells adhere and spread when cultured on top of laminin-coated coverslips or on human amnion basement membrane. M5076 mouse reticulum cell sarcoma cells poorly adhere to these substrates and remain round. Both cell types are invasive in confronting cultures with embryonic chick heart fragments. (+)-Catechin binds to laminin in a pH-dependent way. Pretreatment of laminin-coated coverslips or amnion basement membrane with 0.5 mM (+)-catechin abrogates the effect of laminin on cell morphology and adhesion. MO4 cells do not adhere to the pretreated substrates and remain round, while M5076 cells now adhere and spread. (+)-Catechin inhibits the invasion of MO4 cells but not of M5076 cells into embryonic chick heart in vitro. We speculate that the anti-invasive activity of the flavonoid to MO4 cells is the result of its interference with MO4 cell adhesion to laminin. Invasion of M5076 cells does not imply adhesion to and spreading on laminin.

Pathology of tumours produced in syngeneic Fischer rats by fibroblast-like cells before and after transfection with oncogenes.

Fischer rat cells before and after transfection with immortalizing and transforming genes produced tumours after s.c., i.p., or i.v. injection of cell suspensions and after s.c. implantation of cellular aggregates in the tail of syngeneic rats. Tumours were described histologically as fibrosarcoma-like. Virtually all tumours were considered macroscopically to be invasive because they adhered to the neighbouring tissues; in many tumours invasion was confirmed microscopically. All types of cells produced lung colonies (artificial metastases) after i.v. injection. Spontaneous metastases (from a primary tumour) were found with some tumours produced by cells before as well as after transfection. Differences in metastasis between various cell types could not be ascribed to variations in the periods of observation, in the minimum tumour-bearing periods, in the latency periods, or in the volume of primary tumours. We concluded that local invasion and spontaneous metastasis are usefull for the characterization of malignancy in experimental fibrosarcoma-like tumours. Since Fischer rat cells produced invasive and sometimes metastatic tumours before transfection, the present data do not show a rôle of immortalizing and transforming genes in the acquisition of invasiveness and metastatic capability.