V3 versican isoform alters the behavior of human melanoma cells by interfering with CD44/ErbB-dependent signaling

Versican is a hyaluronan-binding, extracellular chondroitin sulfate proteoglycan produced by several tumor types, including malignant melanoma, which exists as four different splice variants. The short V3 isoform contains the G1 and G3 terminal domains of versican that may potentially interact directly or indirectly with the hyaluronan receptor CD44 and the EGFR, respectively. We have previously described that overexpression of V3 in MeWo human melanoma cells markedly reduces tumor cell growth in vitro and in vivo. In this study we have investigated the signaling mechanism of V3 by silencing the expression of CD44 in control and V3-expressing melanoma cells. Suppression of CD44 had the same effects on cell proliferation and cell migration than those provoked by V3 expression, suggesting that V3 acts through a CD44-mediated mechanism. Furthermore, CD44-dependent hyaluronan internalization was blocked by V3 expression and CD44 silencing, leading to an accumulation of this glycosaminoglycan in the pericellular matrix and to changes in cell migration on hyaluronan. Furthermore, ERK1/2 and p38 activation after EGF treatment were decreased in V3-expressing cells suggesting that V3 may also interact with the EGFR through its G3 domain. The existence of a EGFR/ErbB2 receptor complex able to interact with CD44 was identified in MeWo melanoma cells. V3 overexpression resulted in a reduced interaction between EGFR/ErbB2 and CD44 in response to EGF treatment. Our results indicate that the V3 isoform of versican interferes with CD44 and the CD44-EGFR/ErbB2 interaction, altering the signaling pathways, such as ERK1/2 and p38 MAPK, that regulate cell proliferation and migration.     

LRP-1 silencing prevents malignant cell invasion despite increased pericellular proteolytic activities

The scavenger receptor low-density lipoprotein receptor-related protein 1 (LRP-1) mediates the clearance of a variety of biological molecules from the pericellular environment, including proteinases which degrade the extracellular matrix in cancer progression. However, its accurate functions remain poorly explored and highly controversial. Here we show that LRP-1 silencing by RNA interference results in a drastic inhibition of cell invasion despite a strong stimulation of pericellular matrix metalloproteinase 2 and urokinase-type plasminogen activator proteolytic activities. Cell migration in both two and three dimensions is decreased by LRP-1 silencing. LRP-1-silenced carcinoma cells, which are characterized by major cytoskeleton rearrangements, display atypical overspread morphology with a lack of membrane extensions. LRP-1 silencing accelerates cell attachment, inhibits cell-substrate deadhesion, and induces the accumulation, at the cell periphery, of abundant talin-containing focal adhesion complexes deprived of FAK and paxillin. We conclude that in addition to its role in ligand binding and endocytosis, LRP-1 regulates cytoskeletal organization and adhesive complex turnover in malignant cells by modulating the focal complex composition, thereby promoting invasion.     

Role of LRP-1 in cancer cell migration in 3-dimensional collagen matrix

The low-density lipoprotein receptor-related protein-1 (LRP-1) is a member of Low Density Lipoprotein Receptor (LDLR) family, which is ubiquitously expressed and which is described as a multifunctional endocytic receptor which mediates the clearance of various extracellular matrix molecules including serine proteinases, proteinase-inhibitor complexes, and matricellular proteins. Several studies showed that high LRP-1 expression promotes breast cancer cell invasiveness, and LRP-1 invalidation leads to cell motility abrogation in both tumor and non-tumor cells. Furthermore, our group has reported that LRP-1 silencing prevents the invasion of a follicular thyroid carcinoma despite increased pericellular proteolytic activities from MMP2 and uPA using a 2D-cell culture model. As the use of 3D culture systems is becoming more and more popular due to their promise as enhanced models of tissue physiology, the aim of the present work is to characterize for the first time how the 3D collagen type I matrix may impact the ability of LRP-1 to regulate the migratory properties of thyroid carcinoma using as a model FTC-133 cells. Our results show that inhibition of LRP-1 activity or expression leads to morphological changes affecting cell-matrix interactions, reorganizations of the actin-cytoskeleton especially by inhibiting FAK activation and increasing RhoA activity and MLC-2 phosphorylation, thus preventing cell migration. Taken together, our results suggest that LRP-1 silencing leads to a decrease of cell migratory capacity in a 3D configuration.     

Regulation of CD44 alternative splicing by SRm160 and its potential role in tumor cell invasion

The multiple isoforms of the transmembrane glycoprotein CD44 are produced by alternative RNA splicing. Expression of CD44 isoforms containing variable 5 exon (v5) correlates with enhanced malignancy and invasiveness of some tumors. Here we demonstrate that SRm160, a splicing coactivator, regulates CD44 alternative splicing in a Ras-dependent manner. Overexpression of SRm160 stimulates inclusion of CD44 v5 when Ras is activated. Conversely, small interfering RNA (siRNA)-mediated silencing of SRm160 significantly reduces v5 inclusion. Immunoprecipitation shows association of SRm160 with Sam68, a protein that also stimulates v5 inclusion in a Ras-dependent manner, suggesting that these two proteins interact to regulate CD44 splicing. Importantly, siRNA-mediated depletion of CD44 v5 decreases tumor cell invasion. Reduction of SRm160 by siRNA transfection downregulates the endogenous levels of CD44 isoforms, including v5, and correlates with a decrease in tumor cell invasiveness.     

CD44 interaction with tiam1 promotes Rac1 signaling and hyaluronic acid-mediated breast tumor cell migration

In this study we have explored the interaction between CD44 (the hyaluronic acid (HA)-binding receptor) and Tiam1 (a guanine nucleotide exchange factor) in metastatic breast tumor cells (SP1 cell line). Immunoprecipitation and immunoblot analyses indicate that both the CD44v3 isoform and the Tiam1 protein are expressed in SP1 cells and that these two proteins are physically associated as a complex in vivo. Using an Escherichia coli-derived calmodulin-binding peptide-tagged Tiam1 fragment (i.e. the NH(2)-terminal pleckstrin homology (PHn) domain and an adjacent protein interaction domain designated as PHn-CC-Ex, amino acids 393-738 of Tiam1) and an in vitro binding assay, we have detected a specific binding interaction between the Tiam1 PHn-CC-Ex domain and CD44. Scatchard plot analysis indicates that there is a single high affinity CD44 binding site in the PHn-CC-Ex domain of Tiam1 with an apparent dissociation constant (K(d)) of 0.2 nM, which is comparable with CD44 binding (K(d) = approximately 0.13 nM) to intact Tiam1. These findings suggest that the PHn-CC-Ex domain is the primary Tiam1-binding region for CD44. Most importantly, the binding of HA to CD44v3 of SP1 cells stimulates Tiam1-catalyzed Rac1 signaling and cytoskeleton-mediated tumor cell migration. Transfection of SP1 cells with Tiam1cDNA promotes Tiam1 association with CD44v3 and up-regulates Rac1 signaling as well as HA/CD44v3-mediated breast tumor cell migration. Co-transfection of SP1 cells with PHn-CC-Ex cDNA and Tiam1 cDNA effectively inhibits Tiam1 association with CD44 and efficiently blocks tumor behaviors. Taken together, we believe that the linkage between CD44v3 isoform and the PHn-CC-EX domain of Tiam1 is required for HA stimulated Rac1 signaling and cytoskeleton-mediated tumor cell migration during breast cancer progression.     

A critical role for tetraspanin CD151 in alpha3beta1 and alpha6beta4 integrin-dependent tumor cell functions on laminin-5

The basement membrane protein laminin-5 supports tumor cell adhesion and motility and is implicated at multiple steps of the metastatic cascade. Tetraspanin CD151 engages in lateral, cell surface complexes with both of the major laminin-5 receptors, integrins alpha3beta1 and alpha6beta4. To determine the role of CD151 in tumor cell responses to laminin-5, we used retroviral RNA interference to efficiently silence CD151 expression in epidermal carcinoma cells. Near total loss of CD151 had no effect on steady state cell surface expression of alpha3beta1, alpha6beta4, or other integrins with which CD151 associates. However, CD151-silenced carcinoma cells displayed markedly impaired motility on laminin-5, accompanied by unusually persistent lateral and trailing edge adhesive contacts. CD151 silencing disrupted alpha3beta1 integrin association with tetraspanin-enriched microdomains, reduced the bulk detergent extractability of alpha3beta1, and impaired alpha3beta1 internalization in cells migrating on laminin-5. Both alpha3beta1- and alpha6beta4-dependent cell adhesion to laminin-5 were also impaired in CD151-silenced cells. Reexpressing CD151 in CD151-silenced cells reversed the adhesion and motility defects. Finally, loss of CD151 also impaired migration but not adhesion on substrates other than laminin-5. These data show that CD151 plays a critical role in tumor cell responses to laminin-5 and reveal promotion of integrin recycling as a novel potential mechanism whereby CD151 regulates tumor cell migration.     

Tumor cell programmed death ligand 1-mediated T cell suppression is overcome by coexpression of CD80

Programmed death ligand 1 (PDL1, or B7-H1) is expressed constitutively or is induced by IFN-γ on the cell surface of most human cancer cells and acts as a "molecular shield" by protecting tumor cells from T cell-mediated destruction. Using seven cell lines representing four histologically distinct solid tumors (lung adenocarcinoma, mammary carcinoma, cutaneous melanoma, and uveal melanoma), we demonstrate that transfection of human tumor cells with the gene encoding the costimulatory molecule CD80 prevents PDL1-mediated immune suppression by tumor cells and restores T cell activation. Mechanistically, CD80 mediates its effects through its extracellular domain, which blocks the cell surface expression of PDL1 but does not prevent intracellular expression of PDL1 protein. These studies demonstrate a new role for CD80 in facilitating antitumor immunity and suggest new therapeutic avenues for preventing tumor cell PDL1-induced immune suppression.     

TSG-6 modulates the interaction between hyaluronan and cell surface CD44

Interactions between CD44 and hyaluronan are implicated in the primary adhesion of lymphocytes to endothelium at inflammatory locations. Here we show that preincubation of hyaluronan with full-length recombinant TSG-6 or its Link module domain (Link_TSG6) enhances or induces the binding of hyaluronan to cell surface CD44 on constitutive and inducible cell backgrounds, respectively. These effects are blocked by CD44-specific antibodies and are absent in CD44-negative cells. Enhancement of CD44-mediated interactions of lymphoid cells with hyaluronan by TSG-6 proteins was seen under conditions of flow at shear forces that occur in post-capillary venules. Increases in the number of rolling cells were observed on substrates comprising TSG-6-hyaluronan complexes as compared with a substrate containing hyaluronan alone. In ligand competition experiments, cell surface-bound TSG-6-hyaluronan complexes were more potent than hyaluronan alone in inhibiting cell adhesion to immobilized hyaluronan. Link_TSG6 mutants with impaired hyaluronan binding function had a reduced ability to modulate ligand binding by cell surface CD44. However, some mutants that exhibited close to wild-type hyaluronan binding were found to have either reduced or increased activity, suggesting that some amino acid residues outside of the hyaluronan binding site might be involved in protein self-association, potentially leading to the formation of cross-linked hyaluronan fibers. In turn, cross-linked hyaluronan could increase the binding avidity of CD44 by inducing receptor clustering. The ability of TSG-6 to modulate the interaction of hyaluronan with CD44 has important implications for CD44-mediated cell activity at sites of inflammation, where TSG-6 is expressed.     

Identification and characterization of the acidic pH binding sites for growth regulatory ligands of low density lipoprotein receptor-related protein-1

The type V TGF-beta receptor (TbetaR-V) plays an important role in growth inhibition by IGFBP-3 and TGF-beta in responsive cells. Unexpectedly, TbetaR-V was recently found to be identical to the LRP-1/alpha(2)M receptor; this has disclosed previously unreported growth regulatory functions of LRP-1. Here we demonstrate that, in addition to expressing LRP-1, all cells examined exhibit low affinity but high density acidic pH binding sites for LRP-1 growth regulatory ligands (TGF-beta(1), IGFBP-3, and alpha(2)M(*)). These sites, like LRP-1, are sensitive to receptor-associated protein and calcium depletion but, unlike LRP-1, are also sensitive to chondroitin sulfate and heparin and capable of directly binding ligands, which do not bind to LRP-1. Annexin VI has been identified as a major membrane-associated protein capable of directly binding alpha(2)M(*) at acidic pH. This is evidenced by: 1) structural and Western blot analyses of the protein purified from bovine liver plasma membranes by alpha(2)M(*) affinity column chromatography at acidic pH, and 2) dot blot analysis of the interaction of annexin VI and (125)I-alpha(2)M(*). Cell surface annexin VI is involved in (125)I-TGF-beta(1) and (125)I-alpha(2)M(*) binding to the acidic pH binding sites and (125)I-alpha(2)M(*) binding to LRP-1 at neutral pH as demonstrated by the sensitivity of cells to pretreatment with anti-annexin VI IgG. Cell surface annexin VI is also capable of mediating internalization and degradation of cell surface-bound (125)I-TGF-beta(1) and (125)I-alpha(2)M(*) at pH 6 and of forming ternary complexes with (125)I-alpha(2)M(*) and LRP-1 at neutral pH as demonstrated by co-immunoprecipitation. Trifluoperazine and fluphenazine, which inhibit ligand binding to the acidic pH binding sites, block degradation after internalization of cell surface-bound (125)I-TGF-beta(1) or (125)I-alpha(2)M(*). These results suggest that cell surface annexin VI may function as an acidic pH binding site or receptor and may also function as a co-receptor with LRP-1 at neutral pH.     

HIV-1 promotes intake of Leishmania parasites by enhancing phosphatidylserine-mediated, CD91/LRP-1-dependent phagocytosis in human macrophages

Over the past decade, the number of reported human immunodeficiency virus type-1 (HIV-1)/Leishmania co-infections has risen dramatically, particularly in regions where both diseases are endemic. Although it is known that HIV-1 infection leads to an increase in susceptibility to Leishmania infection and leishmaniasis relapse, little remains known on how HIV-1 contributes to Leishmania parasitaemia. Both pathogens infect human macrophages, and the intracellular growth of Leishmania is increased by HIV-1 in co-infected cultures. We now report that uninfected bystander cells, not macrophages productively infected with HIV-1, account for enhanced phagocytosis and higher multiplication of Leishmania parasites. This effect can be driven by HIV-1 Tat protein and transforming growth factor-beta (TGF-β). Furthermore, we show for the first time that HIV-1 infection increases surface expression of phosphatidylserine receptor CD91/LRP-1 on human macrophages, thereby leading to a Leishmania uptake by uninfected bystander cells in HIV-1-infected macrophage populations. The more important internalization of parasites is due to interactions between the scavenger receptor CD91/LRP-1 and phosphatidylserine residues exposed at the surface of Leishmania. We determined also that enhanced CD91/LRP-1 surface expression occurs rapidly following HIV-1 infection, and is triggered by the activation of extracellular TGF-β. Thus, these results establish an intricate link between HIV-1 infection, Tat, surface CD91/LRP-1, TGF-β, and enhanced Leishmania phosphatidylserine-mediated phagocytosis.     

CD44 cross-linking induces integrin-mediated adhesion and transendothelial migration in breast cancer cell line by up-regulation of LFA-1 (alpha L beta2) and VLA-4 (alpha4beta1)

CD44, a widely expressed cell surface glycoprotein, plays a major role in cell-cell adhesion, cell-substrate interaction, lymphocyte homing, and tumor metastasis. For tumor metastasis to occur through the blood vessel and lymphatic vessel pathway, the tumor cells must first adhere to endothelial cells. Recent studies have shown that high expression of CD44 in certain types of tumors is associated with the hematogenic spread of cancer cells. However, the functional relevance of CD44 to tumor cell metastasis remains unknown. In this study, we investigated the mechanisms of CD44 cross-linking-induced adhesion and transendothelial migration of tumor cells using MDA-MB-435S breast cancer cell line. Breast cancer cells were found to express high levels of CD44. Using flow cytometric analysis and immunofluorescence staining, we demonstrated that cross-linking of CD44 resulted in a marked induction of the expression of lymphocyte function-associated antigen-1 (LFA-1) and very late antigen-4 (VLA-4) by exocytosis. These results were also observed with the Hs578T breast cancer cell line. Furthermore, LFA-1- and VLA-4-mediated adhesion and transendothelial cancer cell migration were also studied. Anti-LFA-1 mAb or anti-VLA-4 mAb alone had no effect on adhesion or transendothelial cancer cell migration, but were able to inhibit both of these functions when added together. This shows that CD44 cross-linking induces LFA-1 and VLA-4 expression in MDA-MB-435S cells and increases integrin-mediated adhesion to endothelial cells, resulting in the transendothelial migration of breast cancer cells. These observations provide direct evidence of a new function for CD44 that is involved in the induction of LFA-1 and VLA-4 expression by exocytosis in MDA-MB-435S cells. Because these induced integrins promote tumor cell migration into the target tissue, it may be possible to suppress this by pharmacological means, and thus potentially cause a reduction in invasive capability and metastasis.     

MicroRNA miR-328 regulates zonation morphogenesis by targeting CD44 expression

Morphogenesis is crucial to initiate physiological development and tumor invasion. Here we show that a microRNA controls zonation morphogenesis by targeting hyaluronan receptor CD44. We have developed a novel system to study microRNA functions by generating constructs expressing pre-miRNAs and mature miRNAs. Using this system, we have demonstrated that expression of miR-328 reduced cell adhesion, aggregation, and migration, and regulated formation of capillary structure. Protein analysis indicated that miR-328 repressed CD44 expression. Activities of luciferase constructs harboring the target site in CD44, but not the one containing mutation, were repressed by miR-328. Zonation morphogenesis appeared in cells transfected by miR-328: miR-328-transfected cells were present on the surface of zonating structures while the control cells stayed in the middle. MiR-328-mediated CD44 actions was validated by anti-CD44 antibody, hyaluronidase, CD44 siRNA, and CD44 expression constructs. In vivo experiments showed that CD44-silencing cells appeared as layers on the surfaces of nodules or zonating structures. Immuno-histochemistry also exhibited CD44-negative cells on the surface layers of normal rat livers and the internal zones of Portal veins. Our results demonstrate that miR-328 targets CD44, which is essential in regulating zonation morphogenesis: silencing of CD44 expression is essential in sealing the zonation structures to facilitate their extension and to inhibit complex expansion.     

CD44: functional relevance to inflammation and malignancy

CD44 is a principal cell surface receptor for hyaluronan, a major component of extracellular matrices. Cells are surrounded by and encounter matrix in vivo, which in turn serves a variety of cell functions through the direct adhesion via their receptors. CD44 communicates cell-matrix interactions into the cell via "outside-in signaling" and has an important role in biological activities. The interaction of CD44 with fragmented hyaluronan on rheumatoid synovial cells induces expression of VCAM-1 and Fas on the cells, which leads to Fas-mediated apoptosis of synovial cells by the interaction of T cells bearing FasL. On the other hand, engagement of CD44 on tumor cells derived from lung cancer reduces Fas expression and Fas-mediated apoptosis, resulting in less susceptibility of the cells to CTL-mediated cytotoxicity through Fas-FasL pathway. Thus, although the CD44-mediated signaling differs among cells and circumstances, we here propose the functional role of CD44 in inflammatory processes and tumor susceptibility and the rational design of future therapeutic strategies including the exploitation of CD44-mediated pathway in vivo.     

Non-small cell lung cancer cyclooxygenase-2-dependent invasion is mediated by CD44

Elevated tumor cyclooxygenase (COX-2) expression is associated with increased angiogenesis, tumor invasion, and suppression of host immunity. We have previously shown that genetic inhibition of tumor COX-2 expression reverses the immunosuppression induced by non-small cell lung cancer (NSCLC). To assess the impact of COX-2 expression in lung cancer invasiveness, NSCLC cell lines were transduced with a retroviral vector expressing the human COX-2 cDNA in the sense (COX-2-S) and antisense (COX-2-AS) orientations. COX-2-S clones expressed significantly more COX-2 protein, produced 10-fold more prostaglandin E(2), and demonstrated an enhanced invasive capacity compared with control vector-transduced or parental cells. CD44, the cell surface receptor for hyaluronate, was overexpressed in COX-2-S cells, and specific blockade of CD44 significantly decreased tumor cell invasion. In contrast, COX-2-AS clones had a very limited capacity for invasion and showed diminished expression of CD44. These findings suggest that a COX-2-mediated, CD44-dependent pathway is operative in NSCLC invasion. Because tumor COX-2 expression appears to have a multifaceted role in conferring the malignant phenotype, COX-2 may be an important target for gene or pharmacologic therapy in NSCLC.     

CD8-mediated type 1 antitumor responses selectively modulate endogenous differentiated and nondifferentiated T cell localization, activation, and function in progressive breast cancer

CD8 T cell-mediated immune responses fall into two distinct types based on effector cell-derived cytokine production. Type I CD8 T cells (Tc1) produce IFN-gamma, whereas type 2 cells (Tc2) secrete IL-4, IL-5, IL-10, and GM-CSF. Using a murine TCR transgenic T cell/breast tumor model, we show that adoptively transferred Ag-specific Tc1 cells are more effective in delaying mammary tumor growth and progression than that of functionally distinct Tc2 cells. Donor Tc1 cells administered 7 days posttumor challenge localized and persisted at sites of primary tumor growth with antitumor responses that were dependent, in part, on effector cell-derived IFN-gamma. Tc1-mediated responses markedly enhanced the appearance and local accumulation of highly differentiated (CD44(high)) CD4 and CD8 endogenous tumor-infiltrating T cells when compared with that of untreated tumor-bearing mice. Conversely, Tc1 cell transfer markedly delayed the appearance of corresponding nondifferentiated (CD44(low)) endogenous T cells. Such cells were acutely activated as defined by coexpression of surface markers associated with TCR engagement (CD69) and early T cell activation (CD25). Moreover, cellular response kinetics appeared to further correlate with the up-regulation of endogenous T cells producing the chemokine IFN-gamma-inducible protein-10 in vivo. This suggested that CD8-mediated type 1 antitumor responses cannot only promote accumulation of distinct endogenous CD4 and CD8 T cell subpopulations, but also facilitate and preferentially modulate their localization kinetics, persistence, states of activation/differentiation, and function within the primary tumor environment at various stages of tumor progression. These studies offer insight into potential mechanisms for enhancing T cell-based immunotherapy in breast cancer.     

Hepatocyte growth factor enhances adhesion of breast cancer cells to endothelial cells in vitro through up-regulation of CD44

For cancer metastasis, tumor cells present in the circulation must first adhere to the endothelium. Integrins play a central role in leukocyte adhesion to the endothelium and subsequent migration into tissues. The majority of tumor cells derived from solid cancers, including breast cancer, do not express integrins. We investigated the mechanisms of adhesion and transendothelial migration of cancer cells using breast carcinoma cell lines. Our results showed the following features of breast cancer cells: (1) HGF stimulated breast cancer cells by up-regulating CD44 expression in a concentration-dependent manner. (2) the maximum level of HGF-induced CD44 up-regulation on breast cancer cell lines occurred within 3 h. (3) HGF-induced up-regulation of CD44 was mediated by the tyrosine kinase signaling pathway. (4) HGF induced CD44-mediated adhesion of tumor cell lines to bone marrow-derived endothelial cells. (5) HGF did not change rolling of breast cancer cell lines on bone marrow-derived endothelial cells, but enhanced firm adhesion of cancer cells on endothelial cells under shear stress conditions. (6) HGF increased transendothelial migration of cancer cells. Our results indicate that HGF stimulates CD44-mediated adhesion of breast cancer cells to bone marrow-derived endothelial cells, which subsequently results in transendothelial migration of tumor cells. These results suggest that CD44 may confer the metastatic properties of breast cancer cells and, therefore, could be used as a target in future molecular cancer therapy.     

Molecular dissection of the human alpha2-macroglobulin subunit reveals domains with antagonistic activities in cell signaling

alpha(2)-Macroglobulin (alpha(2)M) is a plasma protease inhibitor, which reversibly binds growth factors and, in its activated form, binds to low density lipoprotein receptor-related protein (LRP-1), an endocytic receptor with cell signaling activity. Because distinct domains in alpha(2)M are responsible for its various functions, we hypothesized that the overall effects of alpha(2)M on cell physiology reflect the integrated activities of multiple domains, some of which may be antagonistic. To test this hypothesis, we expressed the growth factor carrier site and the LRP-1 recognition domain (RBD) as separate GST fusion proteins (FP3 and FP6, respectively). FP6 rapidly and robustly activated Akt and ERK/MAP kinase in Schwann cells and PC12 cells. This response was blocked by LRP-1 gene silencing or by co-incubation with the LRP-1 antagonist, receptor-associated protein. The activity of FP6 also was blocked by mutating Lys(1370) and Lys(1374), which precludes LRP-1 binding. FP3 blocked activation of Akt and ERK/MAP kinase in response to nerve growth factor-beta (NGF-beta) but not FP6. In PC12 cells, FP6 promoted neurite outgrowth and expression of growth-associated protein-43, whereas FP3 antagonized the same responses when NGF-beta was added. The ability of FP6 to trigger LRP-1-dependent cell signaling in PC12 cells was reproduced by the 18-kDa RBD, isolated from plasma-purified alpha(2)M by proteolysis and chromatography. We propose that the effects of intact alpha(2)M on cell physiology reflect the degree of penetration of activities associated with different domains, such as FP3 and FP6, which may be regulated asynchronously by conformational change and by other regulatory proteins in the cellular microenvironment.     

Coexpression of CD44 variant (v10/ex14) and CD44S in human mammary epithelial cells promotes tumorigenesis

CD44 is the major hyaluronan cell surface receptor and functions as an adhesion molecule in many different cell types, including human breast epithelial cells. The coexpression of certain CD44 variants (CD44v), such as CD44v (v10/ex14), with CD44s (standard form) appears to be closely associated with human breast tumor metastasis. In this study we have established a stable transfection of CD44v (v10/ex14) cDNA into nontumorigenic human breast epithelial cells (HBL100) which contain endogenous CD44s. Our results indicate that coexpression of both CD44v (v10/ex14) and CD44s alters the following important biological properties of these cells: 1) there is a significant reduction in hyaluronic acid (HA)-mediated cell adhesion; 2) there is an increased migration capability in collagen-matrix gel; and 3) these cells constitutively produce certain angiogenic factors and effectively promote tumorigenesis in athymic nude mice. These findings suggest that coexpression of CD44v (v10/ex14) and CD44s may trigger the onset of cell transformation required for breast cancer development. J. Cell. Physiol. 171:152–160, 1997. © 1997 Wiley-Liss, Inc.