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 TGFbeta1, 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.

BackgroundThe L1 cell adhesion molecule (L1CAM) is potentially involved in epithelial-mesenchymal transition (EMT). EMT marker expression is of prognostic significance in non-small cell lung cancer (NSCLC). The relevance of L1CAM for NSCLC is unclear. We investigated the protein expression of L1CAM in a cohort of NSCLC patients. L1CAM protein expression was correlated with clinico-pathological parameters including survival and markers of epithelial-mesenchymal transition.ResultsL1CAM protein expression was found in 25% of squamous cell carcinomas and 24% of adenocarcinomas and correlated with blood vessel invasion and metastasis (p < 0.05). L1CAM was an independent predictor of survival in a multivariate analysis including pT, pN, and pM category, and tumor differentiation grade. L1CAM expression positively correlated with vimentin, beta-catenin, and slug, but inversely with E-cadherin (all p-values < 0.05). E-cadherin expression was higher in the tumor center than in the tumor periphery, whereas L1CAM and vimentin were expressed at the tumor-stroma interface. In L1CAM-negative A549 cells the L1CAM expression was upregulated and matrigel invasion was increased after stimulation with TGF-beta1. In L1CAM-positive SK-LU-1 and SK-LC-LL cells matrigel invasion was decreased after L1CAM siRNA knockdown.ConclusionsA subset of NSCLCs with vessel tropism and increased metastasis aberrantly expresses L1CAM. L1CAM is a novel prognostic marker for NSCLCs that is upregulated by EMT induction and appears to be instrumental for enhanced cell invasion.

Recent work has identified L1CAM (CD171) as a novel marker for human carcinoma progression. Functionally, L1CAM promotes tumor cell invasion and motility, augments tumor growth in nude mice, and facilitates experimental tumor metastasis. These functional features qualify L1 as an interesting target molecule for tumor therapy. Here, we generated a series of novel monoclonal antibodies (mAb) to the L1CAM ectodomain that were characterized by biochemical and functional means. All novel mAbs reacted specifically with L1CAM and not with the closely related molecule CHL1, whereas antibodies to the COOH terminal part of L1CAM (mAb2C2, mAb745H7, pcytL1) showed cross-reactivity. Among the novel mAbs, L1-9.3 was selected and its therapeutic potential was analyzed in various isotype variants in a model of SKOV3ip cells growing i.p. in CD1 nude mice. Only therapy with the IgG2a variant efficiently prolonged survival and reduced tumor burden. This was accompanied by an increased infiltration of F4/80-positive monocytic cells. Clodronate pretreatment of tumor-bearing animals led to the depletion of monocytes and abolished the therapeutic effect of L1-9.3/IgG2a. Expression profiling of tumor-derived mRNA revealed that L1-9.3/IgG2a therapy induced altered expression of cellular genes associated with apoptosis and tumor growth. Our results establish that anti-L1 mAb therapy acts via immunologic and nonimmunologic effector mechanism to block tumor growth. The novel antibodies to L1CAM could become helpful tools for the therapy of L1-positive human carcinomas.

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 b3-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.

Expression of L1 cell adhesion molecule (L1CAM) is associated with poor prognosis in a variety of human carcinomas including breast, ovarian and pancreatic ductal adenocarcinoma (PDAC). Recently we reported that L1CAM induces sustained nuclear factor kappa B (NF-κB) activation by augmenting the autocrine production of interleukin 1 beta (IL-1β), a process dependent on interaction of L1CAM with integrins. In the present study, we demonstrate that transforming growth factor β1 (TGF-β1) treatment of breast carcinoma (MDA-MB231) and PDAC (BxPc3) cell lines induces an EMT (epithelial to mesenchymal transition)-like phenotype and leads to the expression of L1CAM. In MDA-MB231 cells, up-regulation of L1CAM augmented expression of IL-1β and NF-κB activation, which was reversed by depletion of L1CAM, L1CAM-binding membrane cytoskeleton linker protein ezrin, β1-integrin or focal adhesion kinase (FAK). Over-expression of L1CAM not only induced NF-κB activation but also mediated the phosphorylation of FAK and Src. Phosphorylation was not induced in cells expressing a mutant form of L1CAM (L1-RGE) devoid of the integrin-binding site. FAK- and Src-phosphorylation were inhibited by knock-down of various components of the integrin signalling pathway such as β1- and α5-integrins, integrin-linked kinase (ILK), FAK and the phosphoinositide 3-kinase (PI3K) subunit p110β. In summary, these results reveal that during EMT, L1CAM promotes IL-1β expression through a process dependent on integrin signalling and supports a motile and invasive tumour cell phenotype. We also identify important novel downstream effector molecules of the L1CAM-integrin signalling crosstalk that help to understand the molecular mechanisms underlying L1CAM-promoted tumour progression.

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-jB, which was abolished by L1CAM knockdown. We showed that the expression of IL-1b was selectively upregulated by L1-FL, and increased IL-1b levels were instrumental for sustained NF-jB activation. IL-1b production and NF-jB activation were abolished by knockdown of a5-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-jB activation by IL-1b production and autocrine/paracrine signalling. The unravelling of this novel mechanism sheds new light on the important role of L1CAM expression in PDAC cells.

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BackgroundThe L1 cell adhesion molecule (L1CAM) was originally identified as a neural adhesion molecule involved in axon guidance. In many human epithelial carcinomas L1CAM is overexpressed and thereby augments cell motility, invasion and metastasis formation. L1CAM positive carcinomas are associated with bad prognosis. Recent data point out that L1CAM is regulated in a fashion similar to epithelial-mesenchymal transition (EMT). Previous studies have implied the transcription factors Slug and/or β-catenin in L1CAM transcriptional regulation. However, the regulation of human L1CAM expression at the transcriptional level is not well understood.ResultsTo better understand the molecular basis of L1CAM transcriptional regulation, we carried out a detailed characterization of the human L1CAM promoter. We identified two transcription start sites, the first in front of a non-translated exon 0 (promoter 1) and the other next to the first protein-coding exon 1 (promoter 2). Both sites could be verified in endometrial carcinoma (EC) cell lines and appear to be used in a cell-type specific manner. The two identified promoter regions showed activity in luciferase reporter assays. Chromatin-IP analyses confirmed the in silico predicted E-boxes, binding sites for transcription factors Snail and Slug, as well as Lef-1 sites, which are related to β-catenin-mediated transcriptional regulation, in both promoters. Overexpression of β-catenin exclusively augmented activity of promoter 1 whereas Slug enhanced promoter 1 and 2 activity suggesting that both promoters can be active. Overexpression of β-catenin or Slug could upregulate L1CAM expression in a cell-type specific manner.ConclusionsOur results, for the first time, provide evidence that the L1CAM gene has two functionally active promoter sites that are used in a cell-type specific manner. Slug and β-catenin are involved L1CAM transcriptional regulation. Nevertheless, Slug rather than β-catenin levels are correlated with L1CAM expression in EC cell lines. Our findings suggest that the L1CAM transcriptional regulation is more complex than anticipated and this study provides the basis for a better understanding of L1CAM regulation in non-neuronal/tumor cells.