Mina Fogel

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 αvβ5 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 αvβ5-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 αvβ5. This regulatory loop could be relevant for migratory processes under physiological and pathophysiological conditions.

L1 expression as a predictor of progression and survival in patients with uterine and ovarian carcinomas

BACKGROUND Ovarian and uterine carcinomas are the most common cause of cancer-related deaths in gynecological malignant diseases. We aimed to assess whether the L1 adhesion molecule, an important mediator for cell migration for neural and tumour cells, is expressed in these carcinomas. METHODS We investigated L1 expression by immunohistochemistry, RT-PCR, and Western blot analysis of tumour samples. Soluble L1 in the serum was detected by ELISA and immunoprecipitation. FINDINGS We detected the L1 adhesion molecule in ovarian and uterine tumours in a stage-dependent manner. In a retrospective study L1 was found in 46 of 58 ovarian carcinomas and 20 of 72 uterine adenocarcinomas. L1 expression was an excellent predictor of poor outlook (p<0.00001). Patients with L1 positive uterine tumours were at high risk for progression even in the endometrioid-type tumours, which usually have a favourable prognosis. In uterine tumours, expression of L1 in curettage samples enabled us to identify aggressive tumours before the operation. Soluble L1 was specifically detected in serum samples from patients with ovarian and uterine tumours. ADAM10, which was implicated in previous studies as L1 sheddase, was expressed in tumours in which soluble L1 was present in the serum. INTERPRETATION L1 is overexpressed in ovarian and uterine carcinomas and is associated with short survival. L1 can serve as a new marker for prediction of clinical outcome and could be helpful to identify patients with uterine tumours who are at high risk for recurrent disease. L1 expression and cleavage could promote dissemination of tumours by facilitating cell migration.

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‐β‐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.

Interleukin-1β–Driven Inflammation Promotes the Development and Invasiveness of Chemical Carcinogen–Induced Tumors

The role of microenvironment interleukin 1 (IL-1) on 3-methylcholanthrene (3-MCA)-induced carcinogenesis was assessed in IL-1-deficient mice, i.e., IL-1beta(-/-), IL-1alpha(-/-), IL-1alpha/beta(-/-) (double knockout), and mice deficient in the naturally occurring inhibitor of IL-1, the IL-1 receptor antagonist (IL-1Ra). Tumors developed in all wild-type (WT) mice, whereas in IL-1beta-deficient mice, tumors developed slower and only in some of the mice. In IL-1Ra-deficient mice, tumor development was the most rapid. Tumor incidence was similar in WT and IL-1alpha-deficient mice. Histologic analyses revealed fibrotic structures forming a capsule surrounding droplets of the carcinogen in olive oil, resembling foreign body-like granulomas, which appeared 10 days after injection of 3-MCA and persisted until the development of local tumors. A sparse leukocyte infiltrate was found at the site of carcinogen injection in IL-1beta-deficient mice, whereas in IL-1Ra-deficient mice, a dense neutrophilic infiltrate was observed. Treatment of IL-1Ra-deficient mice with recombinant IL-1Ra but not with an inhibitor of tumor necrosis factor abrogated the early leukocytic infiltrate. The late leukocyte infiltrate (day 70), which was dominated by macrophages, was also apparent in WT and IL-1alpha-deficient mice, but was nearly absent in IL-1beta-deficient mice. Fibrosarcoma cell lines, established from 3-MCA-induced tumors from IL-1Ra-deficient mice, were more aggressive and metastatic than lines from WT mice; cell lines from IL-1-deficient mice were the least invasive. These observations show the crucial role of microenvironment-derived IL-1beta, rather than IL-1alpha, in chemical carcinogenesis and in determining the invasive potential of malignant cells.

The Role of Macrophage-Derived IL-1 in Induction and Maintenance of Angiogenesis

Inflammation and angiogenesis are pivotal processes in the progression of many diseases, including malignancies. A hypoxic microenvironment often results in a milieu of proinflammatory and proangiogenic cytokines produced by infiltrating cells. We assessed the role of macrophage-derived hypoxia-associated cytokines in promoting inflammation and angiogenesis. Supernatants of macrophages, stimulated under hypoxia with or without an inflammatory stimulus (LPS), promoted angiogenesis when incorporated into Matrigel plugs. However, neutralization of IL-1 in the supernatants, particularly IL-1β, completely abrogated cell infiltration and angiogenesis in Matrigel plugs and reduced vascular endothelial growth factor (VEGF) levels by 85%. Similarly, supernatants from macrophages of IL-1β knockout mice did not induce inflammatory or angiogenic responses. The importance of IL-1 signaling in the host was demonstrated by the dramatic reduction of inflammatory and angiogenic responses in Matrigel plugs that contained macrophage supernatants from control mice which had been implanted in IL-1 receptor type I knockout mice. Myeloid cells infiltrating into Matrigel plugs were of bone marrow origin and represented the major source of IL-1 and other cytokines/chemokines in the plugs. Cells of endothelial lineage were the main source of VEGF and were recruited mainly from neighboring tissues, rather than from the bone marrow. Using the aortic ring sprouting assay, it was shown that in this experimental system, IL-1 does not directly activate endothelial cell migration, proliferation and organization into blood vessel-like structures, but rather activates infiltrating cells to produce endothelial cell activating factors, such as VEGF. Thus, targeting IL-1β has the potential to inhibit angiogenesis in pathological situations and may be of considerable clinical value.

L1 adhesion molecule (CD 171) in development and progression of human malignant melanoma

The L1 adhesion molecule (CD171) plays an important role in axon guidance and cell migration in the nervous system. In the human, L1 is expressed on tumors derived from neurocrest and on certain carcinomas. We have analyzed immunohistochemically L1 expression on paraffin embedded specimens of acquired melanocytic nevi, primary cutaneous melanomas, and cutaneous and lymph node metastases of malignant melanomas. We found an increase in L1 immunoreactivity in malignant melanomas and metastases of malignant melanomas as compared to acquired melanocytic nevi that was statistically significant (P<0.05). Additionally, a correlation of L1 immunoreactivity with histological data of prognostic value such as Clark level and the expression of alphav-integrins was found. We detected soluble L1 in the conditioned medium of cultivated melanoma cells but only in 1/40 serum samples from a panel of melanoma patients representing various stages of disease. Our findings suggest that the presence of L1 might contribute to tumor progression by promoting cell adhesion and migration.

CD24 is a marker for human breast carcinoma.

CD24 is a small, mucin-type glycosylphosphatidylinositol-linked cell surface molecule expressed by neutrophils, pre B lymphocytes and certain human tumor cell lines. CD24 has been identified as a ligand for P-selectin in both mouse and human cells. We previously reported that the P-selectin-CD24 binding pathway is important for the binding of the breast carcinoma cell line KS to platelets and the rolling of these cells on endothelial P-selectin. In the present study we have analyzed the expression of CD24 on human breast carcinoma cell lines and on fresh breast carcinoma specimens using the CD24-specific antibody ML-5. Our study clearly demonstrates that CD24 is abundantly expressed on cell lines and fresh tissues of breast carcinomas. We find a differential expression of CD24 in breast carcinomas (cytoplasmic pattern) versus benign breast lesions (apical pattern). Moreover, the intensity of CD24 expression increases with the histological grade of the tumor. Thus, CD24 expression might be a useful marker for human breast carcinoma and play a role in facilitating metastasis by the interaction between tumor cells and platelets or endothelial cells.

Antigenic variation in cancer metastasis: immune escape versus immune control

SummaryAntigenic variation in cancer metastasis was observed in a syngeneic murine tumor system consisting of a low metastatic parental tumor line (derived from a methylcholanthrene-induced DBA/2 T lymphoma, Eb), a high metastatic spontaneous variant thereof (ESb), and a low metastatic ‘revertant’ from ESb (ESb-M). All three lines expressed tumor-associated transplantation antigens (TATA) which elicited specific T cell-mediated antitumor immune reactions in the host. The strongest host response was elicited upon intradermal inoculation. It could be followed by (a) the infiltration of the locally growing tumor by host cells, such as lymphocytes and macrophages, (b) the establishment of specific systemic antitumor immunity, (c) the generation of immune cells capable of transferring protective antitumor immunity into a normal syngeneic recipient, and (d) the generation of tumor specific cytotoxic T lymphocytes (CTL).Anti-TATA CTL were used as typing reagents to investigate the stability or variability in the TATA expression by cloned tumor cell lines. Antigenic variability in the TATA expression was seen under various conditions: (a) clone-dependent variation in the sensitivity to anti-TATA CTL lysis upon prolonged growth in tissue culture, (b) qualitative change in the TATA (TATA1 → TATA2) upon successive i.p. transplantation of the parental Eb tumor line and, (c) generation of TATA negative immune escape variants (TATA2 → TATA-) during metastasis formation from a s.c. site.The relative inefficiency of specific immunization procedures against ESb was found to be due to the effective generation of TATA negative variants by this highly metastatic tumor. The balance between immune control and immune escape could be influenced to the advantage of the host by some means, for instance optimizing the route of antitumor-immune sensitization or by infusion of allogeneic but H-2 identical antitumor-immune T cells. Such immune cells recognized the tumor via minor histocompatibility antigens and thus circumvented the need of TATA recognition. Finally, manipulations at the cell surface of the highly malignant ESb tumor such as those introduced in the ESb-M variant were found to dramatically effect its metastatic potential.

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 β3-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.

Up‐regulation of L1CAM is linked to loss of hormone receptors and E‐cadherin in aggressive subtypes of endometrial carcinomas

Endometrial carcinomas (ECs) are classified into type 1 (less aggressive) and type 2 (aggressive) tumours that differ in genetic alterations. So far, reliable immunohistochemical markers that can identify patients with high risk for recurrence are rare. We have defined the expression of L1 cell adhesion molecule (L1CAM), a biomarker previously identified for EC, and compared its expression to oestrogen receptor (ER)/progesterone receptor (PR) and E‐cadherin. We found that L1CAM was absent in normal endometrium and the vast majority of endometrioid ECs (type 1) but was strongly expressed in serous and clear‐cell ECs, considered as type 2. 78/272 cases were identified as L1CAM‐positive endometrioid ECs that were correlated with a poor prognosis. Strikingly, we observed an inverse relationship between L1CAM and ER/PR/E‐cadherin expression in all ECs. In mixed ECs, composed of endometrioid (L1CAM− ER/PR+ E‐cadherin+) and clear‐cell/serous (L1CAM+ ER/PR− E‐cadherin−), both phenotypes were co‐expressed. In some of these cases L1CAM was up‐regulated at the leading edge of the tumour, where ER/PR and E‐cadherin expression were selectively lost. In EC cell lines treated with the epithelial–mesenchymal transition (EMT) inducer TGFβ1, L1CAM and vimentin were strongly up‐regulated, while E‐cadherin expression was reduced. The treatment also resulted in an increased expression of the EMT transcription factor Slug and an enhanced cell invasion. Depletion of Slug by siRNA knockdown prevented both L1CAM up‐regulation and enhanced cell invasion. According to our analysis, we suggest that L1CAM is a novel marker for EMT in ECs and that L1CAM‐typing could identify endometrioid ECs that have type 2‐like features and are at high risk for recurrence. Copyright