Extracellular vesicles from human saliva promote hemostasis by delivering coagulant tissue factor to activated platelets.

Essentials Human salivary extracellular vesicles (EVs) expose coagulant tissue factor (TF). Salivary EVs expose CD24, a ligand of P-selectin. CD24 and coagulant TF co-localize on salivary EVs. TF+ /CD24+ salivary EVs bind to activated platelets and trigger coagulation. SUMMARY: Background Extracellular vesicles (EVs) from human saliva expose coagulant tissue factor (TF). Whether such TF-exposing EVs contribute to hemostasis, however, is unknown. Recently, in a mice model, tumor cell-derived EVs were shown to deliver coagulant TF to activated platelets at a site of vascular injury via interaction between P-selectin glycoprotein ligand-1 (PSGL-1) and P-selectin. Objectives We hypothesized that salivary EVs may deliver coagulant TF to activated platelets via interaction with P-selectin. Methods We investigated the presence of two ligands of P-selectin on salivary EVs, PSGL-1 and CD24. Results Salivary EVs expose CD24 but PSGL-1 was not detected. Immune depletion of CD24-exposing EVs completely abolished the TF-dependent coagulant activity of cell-free saliva, showing that coagulant TF and CD24 co-localize on salivary EVs. In a whole blood perfusion model, salivary EVs accumulated at the surface of activated platelets and promoted fibrin generation, which was abolished by an inhibitory antibody against human CD24. Conclusions A subset of EVs in human saliva expose coagulant TF and CD24, a ligand of P-selectin, suggesting that such EVs may facilitate hemostasis at a site of skin injury where the wound is licked in a reflex action.

Antibody targeting of CD24 efficiently retards growth and influences cytokine milieu in experimental carcinomas.

BACKGROUND: The targeting of cancer stem cells by monoclonal antibodies offers new options for therapy. CD24 is a glycosylphosphatidylinositol-anchored membrane protein with a small protein core and a high level of glycosylation. It is overexpressed in many human carcinomas and is correlated with poor prognosis. CD24 is a marker for pancreatic and ovarian cancer stem cells, whereas breast cancer stem cells are negative for CD24. In cancer cell lines, changes of CD24 expression can alter cellular properties in vitro and tumour growth in vivo. We have shown before that monotherapy with monoclonal antibody (mAb) SWA11 to CD24 effectively retarded tumour growth in xenotransplanted mice. METHODS: Here, we have investigated in more detail the molecular mechanisms of mAb SWA11 therapeutic effects in A549 lung and SKOV3ip ovarian carcinoma models in scid/beige and CD1 mice, respectively. We focused on anti-proliferative, pro-apoptotic, anti-angiogenic and microenvironmental effects of SWA11 mAb treatment. RESULTS: We find that CD24 targeting is associated with changes in tumour cell proliferation and angiogenesis. The treatment lead to increased infiltration of tumour tissues with immune cells suggesting involvement of ADCC. We found that SWA11 mAb treatment strongly altered the intratumoural cytokine microenvironment. The addition of SWA11 mAb to gemcitabine treatment strongly potentiated its anti-cancer efficacy in A549 lung cancer model. CONCLUSION: Our data demonstrate that targeting of CD24 could be beneficial for the anti-cancer treatment combined with standard chemotherapy regimes.

TGF-ß1-dependent L1CAM expression has an essential role in macrophage-induced apoptosis resistance and cell migration of human intestinal epithelial cells.

Patients with chronic inflammatory bowel disease (IBD) have an increased risk to develop colorectal cancer (CRC) particularly after long duration of the disease. Chronic inflammation of the intestinal mucosa is characterized by a marked enrichment of immune cells such as macrophages as well as by high expression of cytokines and growth factors including transforming growth factor-beta 1 (TGF-ß1). The adhesion molecule L1CAM mediates chemoresistance and migration of tumor cells and is elevated in CRC tissues being associated with metastatic spread and poor prognosis for the patients. In this study, we examine the role of TGF-ß1-induced L1CAM expression and macrophages in malignant transformation of intestinal epithelial cells. We demonstrate that TGF-ß1 stimulation leads to a Slug-dependent upregulation of L1CAM expression already in the colonic intestinal epithelial cell line NCM460 thereby enhancing cell motility and apoptosis resistance. Accordingly, NCM460 cells acquired a migratory and apoptosis-resistant phenotype if transfected with L1CAM. Immunohistochemistry of colonic biopsies revealed considerable L1CAM expression in intestinal epithelial cells in tissues from IBD patients but not in normal colonic tissues. Moreover, L1CAM expression increased with duration of disease being associated with the presence of CD33+ macrophages. Coculture with macrophages generated from monocyte colony-stimulating factor (MCSF)-treated monocytes led to the upregulation of Slug and L1CAM in NCM460 cells thereby elevating cell motility and apoptosis resistance. Pharmacological inhibition of TGF-ß1 signalling abolished expression of Slug and L1CAM in cocultured NCM460 cells resulting in decreased cell migration and apoptosis resistance. In conclusion, these data provide new insights into the mechanisms by which IBD promotes malignant transformation of intestinal epithelial cells and underscore the role of L1CAM and macrophages in this scenario.

Prognostic significance of L1CAM in ovarian cancer and its role in constitutive NF-κB activation.

BACKGROUND: Overexpression of L1-cell adhesion molecule (L1CAM) has been observed for various carcinomas and correlates with poor prognosis and late-stage disease. In vitro, L1CAM enhances proliferation, cell migration, adhesion and chemoresistance. We tested L1CAM and interleukin-1 beta (IL-1ß) expression in tumor samples and ascitic fluid from ovarian carcinoma patients to examine its role as a prognostic marker. PATIENTS AND METHODS: We investigated tumor samples and ascitic fluid from 232 serous ovarian carcinoma patients for L1CAM by enzyme-linked immunosorbent assay. L1CAM expression was correlated with pathoclinical parameters and patients' outcome. IL-1ß levels were measured in tumor cell lysates. Ovarian cancer cell lines were analyzed for the contribution of L1CAM to IL-1ß production and nuclear factor 'kappa-light-chain-enhancer' of activated B-cells (NF-κB) activation. RESULTS: We observed that L1CAM-expressing tumors show a highly invasive phenotype associated with restricted tumor resectability at primary debulking surgery and increased lymphogenic spread. Soluble L1CAM proved to be a marker for poor progression-free survival and chemoresistance. In ovarian carcinoma cell lines, the specific knock-down of L1CAM reduces IL-1ß expression and NF-κB activity. CONCLUSIONS: L1CAM expression contributes to the invasive and metastatic phenotype of serous ovarian carcinoma. L1CAM expression and shedding in the tumor microenvironment could contribute to enhanced invasion and tumor progression through increased IL-1ß production and NF-κB activation.

Regulation of transcription factor E2F3a and its clinical relevance in ovarian cancer.

Recently we showed an integral epidermal growth factor receptor (EGFR)-E2F3a signaling path, in which E2F3a was found to be essential in EGFR-mediated proliferation in ovarian cancer cells. The present work evaluates the clinical relevance of this novel axis and of E2F3a itself in a large set of 130 ovarian cancer specimens. For this purpose E2F3a and its counterpart, E2F3b, were measured by RT-PCR and activated EGFR was assessed by immunohistochemistry. When compared with healthy control tissue, both E2F3 isoforms were overexpressed in the cancers, but only E2F3a expression correlated with tumor stage (ρ=0.349, P=0.0001) and residual disease (ρ=0.254, P=0.004). Univariate survival analyses showed E2F3a and activated EGFR to be associated with poor PFS and OS. Furthermore, a strong, positive correlation between activated EGFR and E2F3a expression was shown (P=0.0001). We further identified two EGFR-independent mechanisms that regulate E2F3a expression, namely one, acting by promoter methylation of miR-34a, which by its physical interaction with E2F3a transcripts causes their degradation, and the second based on 6p22 gene locus amplification. MiRIDIAN-based knockdown and induction of miR-34a evidenced a direct regulatory link between miR-34a and E2F3a, and the tumor-suppressive character of miR-34a was documented by its association with improved survival. Although, 6p22 gene locus amplification was detected in a significant number of ovarian cancer specimens, 6p22 ploidy was not relevant in predicting survival. In Cox regression analysis, E2F3a, but not activated EGFR or miR-34a expression, retained independent prognostic significance (PFS: hazards ratio 3.785 (1.326-9.840), P=0.013; OS: hazards ratio 4.651 (1.189-15.572), P=0.013). These clinical findings highlight the relevance of E2F3a in the biology of ovarian cancer. Moreover, identification of EGFR-independent mechanisms in E2F3a control can be helpful in explaining the non-responsiveness of therapeutic EGFR targeting in ovarian cancer.

L1CAM-integrin interaction induces constitutive NF-kappaB activation in pancreatic adenocarcinoma cells by enhancing IL-1beta expression.

L1 cell adhesion molecule (L1CAM) overexpression is often associated with bad prognosis in various human carcinomas. Recent studies also suggest a role of L1CAM in pancreatic ductal adenocarcinomas (PDAC). To further address its contribution, we expressed functional domains of L1CAM in PT45-P1 PDAC cells. We found that L1CAM that is full length (L1-FL), but neither the soluble ectodomain (L1ecto) nor the cytoplasmic part (L1cyt), could enhance cell proliferation or tumour growth in mice. Expression of L1-FL resulted in constitutive activation of NF-kappaB, which was abolished by L1CAM knockdown. We showed that the expression of IL-1beta was selectively upregulated by L1-FL, and increased IL-1beta levels were instrumental for sustained NF-kappaB activation. IL-1beta production and NF-kappaB activation were abolished by knockdown of alpha5-integrin and integrin-linked kinase, but insensitive to depletion of L1CAM cleavage proteinases. Supporting these data, PT45-P1 cells transduced with an L1CAM mutant deficient in integrin binding (L1-RGE) did not support the described L1-FL functions. Our results suggest that membranous L1CAM interacts with RGD-binding integrins, leading to sustained NF-kappaB activation by IL-1beta production and autocrine/paracrine signalling. The unravelling of this novel mechanism sheds new light on the important role of L1CAM expression in PDAC cells.

Sulforaphane targets pancreatic tumour-initiating cells by NF-kappaB-induced antiapoptotic signalling.

BACKGROUND AND AIMS: Emerging evidence suggests that highly treatment-resistant tumour-initiating cells (TICs) play a central role in the pathogenesis of pancreatic cancer. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered to be a novel anticancer agent; however, recent studies have shown that many pancreatic cancer cells are resistant to apoptosis induction by TRAIL due to TRAIL-activated nuclear factor-kappaB (NF-kappaB) signalling. Several chemopreventive agents are able to inhibit NF-kappaB, and favourable results have been obtained--for example, for the broccoli compound sulforaphane-in preventing metastasis in clinical studies. The aim of the study was to identify TICs in pancreatic carcinoma for analysis of resistance mechanisms and for definition of sensitising agents. METHODS: TICs were defined by expression patterns of a CD44(+)/CD24(-), CD44(+)/CD24(+) or CD44(+)/CD133(+) phenotype and correlation to growth in immunodeficient mice, differentiation grade, clonogenic growth, sphere formation, aldehyde dehydrogenase (ALDH) activity and therapy resistance. RESULTS: Mechanistically, specific binding of transcriptionally active cRel-containing NF-kappaB complexes in TICs was observed. Sulforaphane prevented NF-kappaB binding, downregulated apoptosis inhibitors and induced apoptosis, together with prevention of clonogenicity. Gemcitabine, the chemopreventive agents resveratrol and wogonin, and the death ligand TRAIL were less effective. In a xenograft model, sulforaphane strongly blocked tumour growth and angiogenesis, while combination with TRAIL had an additive effect without obvious cytotoxicity in normal cells. Freshly isolated patient tumour cells expressing markers for TICs could be sensitised by sulforaphane for TRAIL-induced cytotoxicity. CONCLUSION: The data provide new insights into resistance mechanisms of TICs and suggest the combination of sulforaphane with TRAIL as a promising strategy for targeting of pancreatic TICs.

L1 cell adhesion molecule (L1CAM) as a pathogenetic factor in endometriosis.

BACKGROUND: Endometriosis is a benign and progressive disease with a high prevalence. Women with endometriosis, especially with atypical endometriosis, have a higher probability for developing ovarian cancer compared with women without endometriosis. The L1 cell adhesion molecule (L1CAM) is over expressed in ovarian and endometrial carcinomas and is associated with a bad prognosis. Here, we have analysed L1CAM expression in endometriosis. METHODS AND RESULTS: In our study with the samples from 79 patients with, and 37 patients without, endometriosis, we found that endometriosis cell lines and short-term cultures of endometrium from women with endometriosis expressed L1CAM at the mRNA and protein level. Quantitative RT-PCR analysis showed that L1CAM was expressed at significantly higher level in the epithelial compartment from patients with endometriosis compared with healthy controls (P = 0.0126). By immunohistochemical staining, 15 of 31 ovarian endometriotic lesions (48%) were shown to have L1CAM-positive staining. Of these 15 L1CAM-positive samples, 13 were atypical endometriotic lesions. Soluble L1 present in the conditioned medium of epithelial endometrium cultures from women with endometriosis was able to stimulate neurite outgrowth as measured in a chicken ganglion assay. CONCLUSIONS: We propose that L1CAM could promote endometriosis development by increasing enervation and aggravation. L1CAM expression is higher in atypical endometriosis compared with normal endometriosis.

The cytoplasmic part of L1-CAM controls growth and gene expression in human tumors that is reversed by therapeutic antibodies.

L1 cell adhesion molecule (L1-CAM) is a transmembrane cell adhesion molecule involved in cell migration and axon guidance in the developing nervous system. L1 is also overexpressed in ovarian and endometrial carcinomas and is associated with a bad prognosis. In carcinoma cell lines, L1 overexpression augments cell motility, tumor growth in mice and induces expression of Erk-dependent genes. Here, we show that a mutation in the cytoplasmic portion of L1 (T1247A, S1248A) abrogates Erk activation, blocks cell migration on extracellular matrix proteins and did not augment tumor growth in non-obese diabetic/severe combined immuno-deficient mice. In cells expressing mutant L1, the induction of Erk-dependent genes such as beta3-integrin, cathepsin-B and several transcription factors is eliminated and the invasive phenotype is abrogated. L1 antibodies showed similar effects. They prevented Erk activation and interfered with the Erk-dependent gene expression pattern. These findings provide a rationale for the mode of action of L1 antibodies and suggest that interference with L1 function could become a valuable target for therapy.

The role of L1-CAM immunohistochemial staining in the diagnosis of abdominal-pelvic cancer of uncertain primary site in women.

OBJECTIVE: The L1 adhesion molecule (L1-CAM,CD171) is over expressed in ovarian and endometrial carcinomas and other tumors derived from the Mullerian tract. Here we evaluated whether L1-CAM could serve as a novel tumor marker for the diagnosis of metastatic abdominal-pelvic cancers of uncertain origin in women. PATIENTS AND METHODS: During a 6-year period we investigated 28 patients with metastatic abdominal or pelvic cancer with uncertain primary-origin. In all these cases a thorough clinical, surgical, pathologic and immunohistochemistry evaluation was performed and correlated to the L1-CAM expression as determined by immunohistochemical staining. RESULTS: In 20 patients where the differential diagnosis was primary ovarian or endometrial cancer and primary or recurrent colon cancer, L1 immunohistochemistry staining allowed or supported the correct diagnosis. In four cases L1 staining allowed the correct diagnosis between breast and ovarian cancer. In two cases vaginal metastases of unknown origin were positive to L1 immunohistochemistry staining implying their mullerian origin and one case each of inguinal lymph node metastases and abdominal wall cancer that were positive for L1-CAM, allowed the correct diagnosis of primary ovarian cancer. In a whole, L1-CAM was of crucial role of delinating the final diagnosis in 17 of the 28 cases described. CONCLUSIONS: L1-CAM, a new tumor marker, was found to be specific for metastatic cancer originating from mullerian origin. Its incorporation into the conventional immunohistochemistry analysis in cases of cancer of unknown primary in women, allows a correct diagnosis and subsequent treatment in the majority of cases with abdominal-pelvic carcinomatosis.

CD24 is a marker of exosomes secreted into urine and amniotic fluid.

Exosomes are small membrane vesicles that are secreted from a variety of cell types into various body fluids including the blood and urine. These vesicles are thought to play a role in cell-cell interactions. CD24 is a small but extensively glycosylated protein linked to the cell surface by means of a glycosyl-phosphatidylinositol anchor. In this study we found that CD24 is present in membrane vesicles characterized as exosomes that were isolated from the urine of normal individuals. CD24 was expressed by both tubule cells and podocytes and treatment of the latter with a cholesterol-extracting agent, but not with a calcium ionophore, caused the release of CD24-containing exosomes. Using CD24 as a marker, we found exosomes in the urine of newborn infants and in the amniotic fluid of pregnant women with similar findings made in mice. Interestingly, studies with CD24 knockout mice showed that the exosomes are released from the fetus but not from the mother; however, exosome release was similar from both the knockout and the wild-type mice. This indicates that CD24 is not essential for exosome formation or release but may be a convenient exosome marker. Our studies suggest that exosomal secretion from the embryonic kidney could play a biological role at the fetal-maternal interphase.

Drug-induced expression of the cellular adhesion molecule L1CAM confers anti-apoptotic protection and chemoresistance in pancreatic ductal adenocarcinoma cells.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by rapid tumor progression, high metastatic potential and profound chemoresistance. We recently reported that induction of a chemoresistant phenotype in the PDAC cell line PT45-P1 by long-term chemotherapy involves an increased interleukin 1 beta (IL1beta)-dependent secretion of nitric oxide (NO) accounting for efficient caspase inhibition. In the present study, we elucidated the involvement of L1CAM, an adhesion molecule previously found in other malignancies, in this NO-dependent chemoresistance. Chemoresistant PT45-P1res cells, but not chemosensitive parental PT45-P1 cells, express high levels of L1CAM in an ILbeta-dependent fashion. PT45-P1res cells subjected to short interfering RNA (siRNA)-mediated L1CAM knock-down exhibited reduced inducible nitric oxide synthase expression and NO secretion, as well as a significant increase of anti-cancer drug-induced caspase activation, an effect reversed by the NO donor S-nitroso-N-acetyl-D,L-penicillamine. Conversely, overexpression of L1CAM in PT45-P1 cells conferred anti-apoptotic protection to anti-cancer drug treatment. Interestingly, L1CAM ectodomain shedding, in example, by ADAM10, as reported for other L1CAM-related activities, seemed to be dispensable for anti-apoptotic protection by L1CAM. Neither the shedded L1CAM ectodomain was detected in chemoresistant L1CAM-expressing PT45-P1 cells nor did the administration of various metalloproteinase inhibitors affect L1CAM-dependent chemoresistance. Immunohistochemical analysis revealed L1CAM expression in 80% of pancreatic cancer specimens, supporting a potential role of L1CAM in the malignancy of this tumor. These findings substantiate our understanding of the molecular mechanisms leading to chemoresistance in PDAC cells and indicate the importance of L1CAM in this scenario.

Different expression levels of alpha3/4 fucosyltransferases and Lewis determinants in ovarian carcinoma tissues and cell lines.

Ovarian carcinoma is the leading cause of death from gynecological cancers in many countries. Fucosylated glycoconjugates have been associated with various carcinomas. In the present study, we have characterized the expression of alpha3/4 fucosyltransferases transcripts and their products, the Lewis carbohydrate determinants, and their in vitro specificity towards synthetic acceptors using ovarian carcinoma cell lines OVM, m130, GG and SKOV3. We found different expression patterns: GG cells expressed mostly Lewisx (Lex), Lewisy (Ley), sLea and Leb, and m130 cells expressed mostly Lex and Ley. The detection was on the plasma membrane and in intracellular vesicles. OVM and SKOV3 cells had very low amounts of staining. From RT-PCR studies, enzyme specificity of cellular extracts towards a panel of synthetic carbohydrate acceptors and Western blot analysis we concluded that Lea, sLea and Leb were synthesised by FUT3, whereas Lex and Ley were synthesized by FUT4 and FUT9 in both cell lines. The GG and m130 cell lines are adequate models to investigate the role of Lex, Ley, sLea and Leb in ovarian carcinoma development.

The role of L1 in the progression of ovarian carcinomas.

The L1 molecule has recently emerged as a promising new biomarker for the prognosis of human ovarian and endometrial tumors. It was initially described as an adhesion molecule for the development of the nervous system but its function in tumor cells is not well known. In this article we summarize recent data on the role of L1 in promoting tumor cell adhesion and migration and in gene regulation. We address the question how L1 determination in tumor tissue samples and in serum and ascites could potentially improve the disease management.

Tumour biological aspects of CD24, a mucin-like adhesion molecule.

CD24 is a molecule that recently has raised considerable attention in tumour biology. It is involved in cell adhesion and metastatic tumour spread. It has also been described as a new diagnostic marker of tumours, of neuroendocrine differentiation and, possibly most intriguing of all, of patient prognosis. High rates of CD24 expression detected by immunohistochemistry have been found in epithelial ovarian cancer (83%), breast cancer (85%), non-small cell lung cancer (45%), prostate cancer (48%) and pancreatic cancer (72%). With the exception of pancreatic cancer, high rates of CD24 are significantly associated with a more aggressive course of the disease, a finding that remains significant in a multivariate analysis. The aim of this review is to summarize relevant work covering these aspects of CD24.

N-glycosylation patterns of HSA/CD24 from different cell lines and brain homogenates: a comparison.

N-glycans of the mouse glycoprotein HSA and its human analogue CD24 from lymphoblastoma, neuroblastoma and astrocytoma cell lines as well as from mouse brain homogenate were analysed and compared to each other and to the N-glycosylation pattern of total glycoproteins from mouse and human brain. The N-glycans were released from PVDF-blotted HSA or CD24 and separated on Carbograph SPE into neutral and acid glycans. The naturally neutral glycan fraction and the fraction of glycans rendered neutral after neuraminidase treatment were analysed without further purification by MALDI-MS. In each fraction, about 25 molecular ions with an intensity >10% of the base peak were identified which corresponded to glycans with distinct isobaric monosaccharide compositions. Comparison of the neutral and desialylated glycans revealed some similarities between the samples analysed, but also clear differences. HSA and CD24 from all cell lines express almost no neutral N-glycans with two or more fucose in contrast to brain HSA and glycoproteins from mouse and human brain. The lack of extensive fucosylation was also observed for desialylated glycans of HSA and CD24 from all cell lines analysed except for CD24 from a human neuroblastoma cell line which exhibits like total human and mouse brain glycoproteins a large variety of highly fucosylated, higher branched N-glycans. HSA from mouse brain carries in addition desialylated non-fucosylated glycans of high abundance which were detected, if at all, only at low intensity in all other samples analysed suggesting that they may be implicated in specific functions of mouse brain HSA. Therefore, a rapid assessment of similarities or differences between glycosylation patterns of a glycoprotein isolated from different sources is possible using methods as described here.

Ectodomain shedding of L1 adhesion molecule promotes cell migration by autocrine binding to integrins.

The L1 adhesion molecule plays an important role in axon guidance and cell migration in the nervous system. L1 is also expressed by many human carcinomas. In addition to cell surface expression, the L1 ectodomain can be released by a metalloproteinase, but the biological function of this process is unknown. Here we demonstrate that membrane-proximal cleavage of L1 can be detected in tumors and in the developing mouse brain. The shedding of L1 involved a disintegrin and metalloproteinase (ADAM)10, as transfection with dominant-negative ADAM10 completely abolishes L1 release. L1-transfected CHO cells (L1-CHO) showed enhanced haptotactic migration on fibronectin and laminin, which was blocked by antibodies to alpha v beta 5 and L1. Migration of L1-CHO cells, but not the basal migration of CHO cells, was blocked by a metalloproteinase inhibitor, indicating a role for L1 shedding in the migration process. CHO and metalloproteinase-inhibited L1-CHO cells were stimulated to migrate by soluble L1-Fc protein. The induction of migration was blocked by alpha v beta 5-specific antibodies and required Arg-Gly-Asp sites in L1. A 150-kD L1 fragment released by plasmin could also stimulate CHO cell migration. We propose that ectodomain-released L1 promotes migration by autocrine/paracrine stimulation via alpha v beta 5. This regulatory loop could be relevant for migratory processes under physiological and pathophysiological conditions.

Critical amino acid residues of the alpha4 subunit for alpha4beta7 integrin function.

A characteristic feature of integrin-ligand interactions is the requirement for divalent cations. Putative cation binding sites have been identified in the alpha and beta subunit of the alpha4 integrins, alpha4beta1 and alpha4beta7, and within their ligands which display the tripeptide LDV in fibronectin and homologous motifs in VCAM-1 and MAdCAM-1. The extracellular domain of the murine and human alpha4-subunit contains three conserved LDV motifs, designated LDV-1 to -3. Using site directed mutagenesis and transfection studies, we now examined the functional relevance of the LDV motifs for alpha4beta7 integrins. We present evidence that LDV-1 mutants (D489N) behave like alpha4 wt cells, but LDV-3 mutants (D811N) are impaired in alpha4beta7 integrin-triggered homotypic cell aggregation and in adhesion and spreading on alpha4 specific ligands. Further characterization of LDV-3 mutants revealed a defect in mAb-induced alpha4beta7-cell surface cluster formation. Mutation of the LDV-2 motif (D698N) caused loss of alpha4beta7 integrin cell surface expression. Our results indicate: (i) that LDV-3, located proximal to the cell membrane, is important for alpha4beta7 integrin-triggered functions and for lateral clustering and (ii) that LDV-2 affects alpha4beta7 heterodimer stability.

Tumor cell invasion is promoted by activation of protease activated receptor-1 in cooperation with the alpha vbeta 5 integrin.

The first prototype of the protease activated receptor (PAR) family, the thrombin receptor PAR1, plays a central role both in the malignant invasion process of breast carcinoma metastasis and in the physiological process of placental implantation. The molecular mechanism underlying PAR1 involvement in tumor invasion and metastasis, however, is poorly defined. Here we show that PAR1 increases the invasive properties of tumor cells primarily by increased adhesion to extracellular matrix components. This preferential adhesion is accompanied by the cytoskeletal reorganization of F-actin toward migration-favoring morphology as detected by phalloidin staining. Activation of PAR1 increased the phosphorylation of focal adhesion kinase and paxillin, and the induced formation of focal contact complexes. PAR1 activation affected integrin cell-surface distribution without altering their level of expression. The specific recruitment of alpha(v)beta(5) to focal contact sites, but not of alpha(v)beta(3) or alpha(5)beta(1), was observed by immunofluorescent microscopy. PAR1 overexpressing cells showed selective reciprocal co-precipitation with alpha(v)beta(5) and paxillin but not with alpha(v)beta(3) that remained evenly distributed under these conditions. This co-immunoprecipitation failed to occur in cells containing the truncated form of PAR1 that lacked the entire cytoplasmic portion of the receptor. Thus, the PAR1 cytoplasmic tail is essential for conveying the cross-talk and recruiting the alpha(v)beta(5) integrin. While PAR1 overexpressing cells were invasive in vitro, as reflected by their migration through a Matrigel barrier, invasion was further enhanced by ligand activation of PAR1. Moreover, the application of anti-alpha(v)beta(5) antibodies specifically attenuated this PAR1 induced invasion. We propose that the activation of PAR1 may lead to a novel cooperation with the alpha(v)beta(5) integrin that supports tumor cell invasion.

The CD24/P-selectin binding pathway initiates lung arrest of human A125 adenocarcinoma cells.

Carbohydrates on tumor cells have been shown to play an important role in tumor metastasis. We demonstrated before that CD24, a Mr 35,000-60,000 mucine-type glycosylphosphatidylinositol-linked cell surface molecule, can function as ligand for P-selectin and that the sialylLex carbohydrate is essential for CD24-mediated rolling of tumor cells on P-selectin. To investigate the role of both antigens more closely, we transfected human A125 adenocarcinoma cells with CD24 and/or fucosyltransferase VII (Fuc TVII) cDNAs. Stable transfectants expressed CD24 and/or sialylLex. Biochemical analysis confirmed that in A125-CD24/FucTVII double transfectants, CD24 was modified with sialylLex. Only double transfectants showed rolling on P-selectin in vivo. When injected into mice, double transfectants arrested in the lungs, and this step was P-selectin dependent because it was strongly enhanced in lipopolysaccharide (LPS) pretreated wild-type mice but not in P-selectin knockout mice. CD24 modified by sialylLex was required on the tumor cells because the LPS-induced lung arrest was abolished by removal of CD24 from the cell surface by phosphatidylinositol-specific phospholipase C. A125-FucTVII single transfectants expressing sialylLex but not CD24 did not show P-selectin-mediated lung arrest. The sialylLex epitope is abundantly expressed on human carcinomas, and significant correlations between sialylLex expression and clinical prognosis exist. Our data suggest an important role for sialylLex-modified CD24 in the lung colonization of human tumors.