Nuclear translocation and signalling of L1-CAM in human carcinoma cells requires ADAM10 and presenilin/gamma-secretase activity.

L1-CAM (L1 cell-adhesion molecule), or more simply L1, plays an important role in the progression of human carcinoma. Overexpression promotes tumour-cell invasion and motility, growth in nude mice and tumour metastasis. It is feasible that L1-dependent signalling contributes to these effects. However, little is known about its mechanism in tumour cells. We reported previously that L1 is cleaved by ADAM (a disintegrin and metalloprotease) and that the cytoplasmic part is essential for L1 function. Here we analysed more closely the role of proteolytic cleavage in L1-mediated nuclear signalling. Using OVMz carcinoma cells and L1-transfected cells as a model, we found that ADAM10-mediated cleavage of L1 proceeds in lipid raft and non-raft domains. The cleavage product, L1-32, is further processed by PS (presenilin)/gamma-secretase to release L1-ICD, an L1 intracellular domain of 28 kDa. Overexpression of dominant-negative PS1 or use of a specific gamma-secretase inhibitor leads to an accumulation of L1-32. Fluorescence and biochemical analysis revealed a nuclear localization for L1-ICD. Moreover, inhibition of ADAM10 and/or gamma-secretase blocks nuclear translocation of L1-ICD and L1-dependent gene regulation. Overexpression of recombinant L1-ICD mediates gene regulation in a similar manner to full-length L1. Our results establish for the first time that regulated proteolytic processing by ADAM10 and PS/gamma-secretase is essential for the nuclear signalling of L1 in human carcinoma cell lines.

alpha5-integrin is crucial for L1CAM-mediated chemoresistance in pancreatic adenocarcinoma.

We recently showed that the adhesion molecule L1CAM (CD171) is overexpressed in pancreatic adenocarcinoma (PDAC) essentially contributing to chemoresistance of PDAC cells. In search of the mechanisms of this effect we now identified alpha5-integrin as the L1CAM ligand being essential for L1CAM-mediated chemoresistance of these highly malignant tumor cells. Thus, blockade or knock-down of alpha5-integrin in the L1CAM expressing PDAC cell lines PT45-P1res, Colo357 and Panc1 increased anti-cancer drug sensitivity. In line with the previously reported NO-dependent caspase inhibition resulting from L1CAM induced iNOS expression, the loss of chemoresistance upon alpha5-integrin inhibition was preceded by decreased iNOS expression and enhanced caspase-3/-7 activation. Accordingly, the loss of anti-cancer drug protection by alpha5-integrin inhibition could be overcome by administration of the NO-donor SNAP. Moreover, the gain of chemoresistance of parental PT45-P1 cells when transfected with L1CAM was abrogated by alpha5-integrin inhibition, whereas transfection of PT45-P1 cells with an integrin binding-deficient L1CAM mutant (L1mutRGE) did neither induce chemoresistance or iNOS expression nor conferred sensitivity to alpha5-integrin inhibition as seen upon transfection with wild-type L1CAM. Thus, mutational loss of the integrin binding site in the L1CAM molecule or the blockade of alpha5-integrin abolished the induction of iNOS expression and chemoresistance by L1CAM, indicating that both a functional L1CAM and alpha5-integrin are indispensable of L1CAM-induced drug resistance in PDAC cells.

The RGD integrin binding site in human L1-CAM is important for nuclear signaling.

L1 cell adhesion molecule (L1-CAM) is a transmembrane cell adhesion molecule initially defined as a promigratory molecule in the developing nervous system. L1 is also overexpressed in a variety of human carcinomas and is associated with bad prognosis. In carcinoma cell lines L1 augments cell motility and metastasis, tumor growth in nude mice and induces expression of L1-dependent genes. It is not known whether L1-signaling requires ligand binding. The RGD motif in the sixth Ig domain of L1 is a binding site for integrins. In the present study we analyzed the role of RGDs in L1-signaling using site-directed mutagenesis combined with antibody blocking studies. We observed that L1-RGE expressing HEK293 cells showed reduced cell-cell binding, cell motility, invasiveness and tumor growth in NOD/SCID mice. The RGE-mutation impaired L1-dependent gene regulation and antibodies to alphavbeta5 integrin had similar effects. Mutant L1 was unable to translocate to the nucleus. Our findings highlight the importance of the RGD site in L1 for human tumors and suggest that nuclear signaling of L1 is dependent on integrins.

L1-CAM in a membrane-bound or soluble form augments protection from apoptosis in ovarian carcinoma cells.

OBJECTIVE: Apoptosis resistance is a hallmark of cancer progression, a phenomenon frequently observed in ovarian carcinoma. We reported previously, that L1 adhesion molecule (CD171) is overexpressed in ovarian and endometrial carcinomas and that L1 expression is a predictor of poor outcome. We investigated a possible role of L1 in apoptosis resistance. METHODS: We used L1 transfectants and ovarian carcinoma cell lines and induced apoptosis by different stimuli such as C2-ceramide, staurosporine, cisplatin or hypoxia. RESULTS: We found that cells expressing L1 are more resistant against apoptosis. In HEK293 cells, L1-expresssion leads to a sustained ERK, FAK and PAK phosphorylation. Soluble L1 only partially rescued HEK293 cells from apoptosis. Treatment with apoptotic stimuli upregulated the anti-apoptotic molecule Bcl-2 to a greater extend in HEK293 cells expressing L1. In the ovarian carcinoma cell line OVMz, the depletion of L1 by RNA interference sensitized cells for apoptosis induction. No changes in activation of ERK or FAK were observed after L1 knockdown. The selection of m130 ovarian carcinoma or SW707 colon carcinoma cells with cisplatin leads to upregulated expression of L1. CONCLUSIONS: Our results suggest a link between L1 expression and chemoresistance of ovarian carcinomas. Upregulation of L1 after cisplatin treatment might indicate a more malignant tumor phenotype given the established role of L1 in cell motility and invasion.

The adhesion molecule L1 (CD171) promotes melanoma progression.

The adhesion molecule L1 is expressed in primary melanomas and cutaneous metastases in contrast to melanocytic nevi and melanocytes, and is significantly associated with metastatic spread. Recent studies have demonstrated that in carcinomas L1 expression is associated with sustained activation of the extracellular signal-regulated kinase (ERK) pathway and upregulation of ERK-dependent, motility- and invasion-associated gene products including alphavbeta3 integrin. The objective of this study was to further investigate the role of the adhesion molecule L1 in melanoma progression, and to evaluate whether targeting the L1 adhesion molecule would have therapeutic effects against invasive melanoma growth. Using human melanoma cells from different stages of progression in monolayer and organotypic human skin culture mimicking the pathophysiological environment of cutaneous melanoma, we found that (1) L1 expression mostly correlates with melanoma progression and alphavbeta3 integrin expression, (2) overexpression of L1 in early radial growth phase melanoma cells promotes conversion from radial to vertical growth phase melanoma without upregulation of alphavbeta3 integrin expression, and (3) suppression of L1 function significantly reduces migration and invasion of melanoma cells, but does not completely block invasive melanoma growth. Altogether, L1 plays a critical role in melanoma invasion and progression and offers therapeutic potential in combination with conventional anticancer agents.

Efficient inhibition of intra-peritoneal tumor growth and dissemination of human ovarian carcinoma cells in nude mice by anti-L1-cell adhesion molecule monoclonal antibody treatment.

The L1 cell adhesion molecule is implicated in the control of proliferation, migration, and invasion of several tumor cell types in vitro. Recently, L1 overexpression was found to correlate with tumor progression of ovarian carcinoma, one of the most common causes of cancer-related deaths in gynecologic malignant diseases. To evaluate L1 as a potential target for ovarian cancer therapy, we investigated the effects of anti-L1 monoclonal antibodies (chCE7 and L1-11A) on proliferation and migration of L1-positive human SKOV3ip ovarian carcinoma cells in vitro and the therapeutic efficacy of L1-11A against i.p. SKOV3ip tumor growth in nude mice. In vitro, both anti-L1 antibodies efficiently inhibited the proliferation of SKOV3ip cells as well as other L1-expressing tumor cell lines (renal carcinoma, neuroblastoma, and colon carcinoma). On two cell lines, hyper-cross-linking of L1-11A with a secondary antibody was necessary for significant inhibition of proliferation, indicating that cross-linking of L1 is required for the antiproliferative effect. L1-negative prostate carcinoma cells were not influenced by antibody treatment. Biweekly treatment of ovarian carcinoma-bearing mice with L1-11A led to a dose-dependent and significant reduction of tumor burden (up to -63.5%) and ascites formation (up to -75%). This effect was associated with reduced proliferation within the tumors. L1-directed antibody-based inhibition of peritoneal growth and dissemination of human ovarian carcinoma cells represents important proof-of-principle for the development of a new therapy against one of the leading gynecologic malignant diseases.

Cleavage of L1 in exosomes and apoptotic membrane vesicles released from ovarian carcinoma cells.

PURPOSE: The L1 adhesion molecule (CD171) is overexpressed in human ovarian and endometrial carcinomas and is associated with bad prognosis. Although expressed as a transmembrane molecule, L1 is released from carcinoma cells in a soluble form. Soluble L1 is present in serum and ascites of ovarian carcinoma patients. We investigated the mode of L1 cleavage and the function of soluble L1. EXPERIMENTAL DESIGN: We used ovarian carcinoma cell lines and ascites from ovarian carcinoma patients to analyze soluble L1 and L1 cleavage by Western blot analysis and ELISA. RESULTS: We find that in ovarian carcinoma cells the constitutive cleavage of L1 proceeds in secretory vesicles. We show that apoptotic stimuli like C2-ceramide, staurosporine, UV irradiation, and hypoxic conditions enhance L1-vesicle release resulting in elevated levels of soluble L1. Constitutive cleavage of L1 is mediated by a disintegrin and metalloproteinase 10, but under apoptotic conditions multiple metalloproteinases are involved. L1 cleavage occurs in two types of vesicles with distinct density features: constitutively released vesicles with similarity to exosomes and apoptotic vesicles. Both types of L1-containing vesicles are present in the ascites fluids of ovarian carcinoma patients. Soluble L1 from ascites is a potent inducer of cell migration and can trigger extracellular signal-regulated kinase phosphorylation. CONCLUSIONS: We suggest that tumor-derived vesicles may be an important source for soluble L1 that could regulate tumor cell function in an autocrine/paracrine fashion.

Glioblastoma-induced attraction of endogenous neural precursor cells is associated with improved survival.

Neural precursor cells contribute to adult neurogenesis and to limited attempts of brain repair after injury. Here we report that in a murine experimental glioblastoma model, endogenous neural precursors migrate from the subventricular zone toward the tumor and surround it. The association of endogenous precursors with syngenic tumor grafts was observed, after injecting red fluorescent protein-labeled G261 cells into the caudate-putamen of transgenic mice, which express green fluorescent protein under a promoter for nestin (nestin-GFP). Fourteen days after inoculation, the nestin-GFP cells surrounded the tumors in several cell layers and expressed markers of early noncommitted and committed precursors. Nestin-GFP cells were further identified by a characteristic membrane current pattern as recorded in acute brain slices. 5-bromo-2-deoxyuridine labeling and dye tracing experiments revealed that the tumor-associated precursors originated from the subventricular zone. Moreover, in cultured explants from the subventricular zone, the neural precursors showed extensive tropism for glioblastomas. Tumor-induced endogenous precursor cell accumulation decreased with age of the recipient; this correlated with increased tumor size and shorter survival times in aged mice. Coinjection of glioblastoma cells with neural precursors improved the survival time of old mice to a level similar to that in young mice. Coculture experiments showed that neural precursors suppressed the rapid increase in tumor cell number, which is characteristic of glioblastoma, and induced glioblastoma cell apoptosis. Our results indicate that tumor cells attract endogenous precursor cells; the presence of precursor cells is antitumorigenic; and this cellular interaction decreases with aging.

L1 augments cell migration and tumor growth but not beta3 integrin expression in ovarian carcinomas.

L1 is a neural cell adhesion molecule involved in cell migration, axon growth and guidance. Recent data have shown that L1 is overexpressed in ovarian and endometrial tumors and is associated with bad prognosis. How L1 promotes tumor progression is presently unknown. Here we show that L1 expression is predominantly confined to the invasive front of ovarian carcinomas. Overexpression of L1 in carcinoma cell lines by adenovirus-mediated gene transfer enhanced the haptotactic cell migration on extracellular matrix proteins. Expression of L1 augmented tumor growth of carcinomas xenografted in nonobese diabetic/severe combined immunodeficient mice (NOD/SCID). A recent report has demonstrated L1-dependent upregulation of beta3 integrin involving activation of the extracellular signal-regulated kinase (erk) pathway. We find that L1 and beta3 integrin are not coexpressed in ovarian carcinoma tissues. Overexpression of L1 did not upregulate beta3 integrin in ovarian carcinoma cell lines but could do so in HEK293 cells. Our results suggest that L1 could drive progression by enhancing cell migration and tumor growth but that L1 dependent and erk-regulated gene expression requires cell-type specific elements.

L1, a novel target of beta-catenin signaling, transforms cells and is expressed at the invasive front of colon cancers.

Aberrant beta-catenin-TCF target gene activation plays a key role in colorectal cancer, both in the initiation stage and during invasion and metastasis. We identified the neuronal cell adhesion molecule L1, as a target gene of beta-catenin-TCF signaling in colorectal cancer cells. L1 expression was high in sparse cultures and coregulated with ADAM10, a metalloprotease involved in cleaving and shedding L1's extracellular domain. L1 expression conferred increased cell motility, growth in low serum, transformation and tumorigenesis, whereas its suppression in colon cancer cells decreased motility. L1 was exclusively localized in the invasive front of human colorectal tumors together with ADAM10. The transmembrane localization and shedding of L1 by metalloproteases could be useful for detection and as target for colon cancer therapy.

Early determination and long-term persistence of adult-generated new neurons in the hippocampus of mice.

New neurons are continually generated in the adult hippocampus, but the important question, whether adult neurogenesis is transient or leads to the lasting presence of new neurons, has not yet been answered. Dividing cells were labeled with bromodeoxyuridine (BrdU) and were investigated by means of immunofluorescence and confocal microscopy at several time-points 1 day to 11 months thereafter. BrdU-labeled neurons remained stable in number and in their relative position in the granule cell layer over at least 11 months. This finding implies that the addition of new neurons is not transient and that their final number and localization are determined early. By contrast, expression of immature markers beta-III-tubulin and doublecortin in BrdU-labeled cells, peaked early after division and was not detectable after 4 weeks. In transgenic mice expressing enhanced green fluorescent protein under the nestin promoter none of the BrdU/nestin-positive cells early after division expressed the mature marker NeuN, confirming that no dividing neurons were detected. These new data suggest that new neurons are recruited early from the pool of proliferating progenitor cells and lead to a lasting effect of adult neurogenesis.

ADAM10-mediated cleavage of L1 adhesion molecule at the cell surface and in released membrane vesicles.

Cells can release membrane components in a soluble form and as membrane vesicles. L1, an important molecule for cell migration of neural and tumor cells, is released by membrane-proximal cleavage, and soluble L1 promotes cell migration. Release of L1 is enhanced by shedding inducers such as phorbol ester and pervanadate, but it is also enhanced by depletion of cellular cholesterol with methyl-beta-cyclodextrin (MCD). How such different compounds can induce shedding is presently unknown. We show here that ADAM10 is involved in L1 cleavage, which occurs at the cell surface and in the Golgi apparatus. MCD and pervanadate treatment induced the release of microvesicles containing full-length L1 and the active form of ADAM10. L1 cleavage occurred in isolated vesicles. L1-containing microvesicles could trigger haptotactic cell migration. Only the neural L1 form carrying the RSLE signal for clathrin-dependent endocytosis was recruited and cleaved in vesicles. Phorbol ester treatment activated L1 cleavage predominantly at the cell surface. Our results provide evidence for two pathways of L1 cleavage, based on ADAM10 localization, that can be activated differentially: 1) direct cleavage at the cell surface, and 2) release and cleavage in secretory vesicles most likely derived from the Golgi apparatus. The findings establish a novel role for ADAM10 as a vesicle-based protease.

Neuroplasticity in old age: sustained fivefold induction of hippocampal neurogenesis by long-term environmental enrichment.

Neurons are continually born from endogenous stem cells and added to the dentate gyrus throughout life, but adult hippocampal neurogenesis declines precipitously with age. Short-term exposure to an enriched environment leads to a striking increase in new neurons, along with a substantial improvement in behavioral performance. Could this plastic response be relevant for explaining the beneficial effects of leading "an active life" on brain function and pathology? Adult hippocampal neurogenesis in mice living in an enriched environment from the age of 10 to 20 months was fivefold higher than in controls. Relatively, the increase in neuronal phenotypes was entirely at the expense of newly generated astrocytes. This cellular plasticity occurred in the context of significant improvements of learning parameters, exploratory behavior, and locomotor activity. Enriched living mice also had a reduced lipofuscin load in the dentate gyrus, indicating decreased nonspecific age-dependent degeneration. Therefore, in mice signs of neuronal aging can be diminished by a sustained active and challenging life, even if this stimulation started only at medium age. Activity exerts not only an acute but also a sustained effect on brain plasticity.