An NKX-COUP-TFII morphogenetic code directs mucosal endothelial addressin expression.

Immunoglobulin family and carbohydrate vascular addressins encoded by Madcam1 and St6gal1 control lymphocyte homing into intestinal tissues, regulating immunity and inflammation. The addressins are developmentally programmed to decorate endothelial cells lining gut post-capillary and high endothelial venules (HEV), providing a prototypical example of organ- and segment-specific endothelial specialization. We identify conserved NKX-COUP-TFII composite elements (NCCE) in regulatory regions of Madcam1 and St6gal1 that bind intestinal homeodomain protein NKX2-3 cooperatively with venous nuclear receptor COUP-TFII to activate transcription. The Madcam1 element also integrates repressive signals from arterial/capillary Notch effectors. Pan-endothelial COUP-TFII overexpression induces ectopic addressin expression in NKX2-3+ capillaries, while NKX2-3 deficiency abrogates expression by HEV. Phylogenetically conserved NCCE are enriched in genes involved in neuron migration and morphogenesis of the heart, kidney, pancreas and other organs. Our results define an NKX-COUP-TFII morphogenetic code that targets expression of mucosal vascular addressins.

A molecular map of murine lymph node blood vascular endothelium at single cell resolution.

Blood vascular endothelial cells (BECs) control the immune response by regulating blood flow and immune cell recruitment in lymphoid tissues. However, the diversity of BEC and their origins during immune angiogenesis remain unclear. Here we profile transcriptomes of BEC from peripheral lymph nodes and map phenotypes to the vasculature. We identify multiple subsets, including a medullary venous population whose gene signature predicts a selective role in myeloid cell (vs lymphocyte) recruitment to the medulla, confirmed by videomicroscopy. We define five capillary subsets, including a capillary resident precursor (CRP) that displays stem cell and migratory gene signatures, and contributes to homeostatic BEC turnover and to neogenesis of high endothelium after immunization. Cell alignments show retention of developmental programs along trajectories from CRP to mature venous and arterial populations. Our single cell atlas provides a molecular roadmap of the lymph node blood vasculature and defines subset specialization for leukocyte recruitment and vascular homeostasis.

Patterns of expression of factor VIII and von Willebrand factor by endothelial cell subsets in vivo.

UNLABELLED: Circulating factor VIII (FVIII) is derived from liver and from extrahepatic sources probably of endothelial origin, but the vascular sites of FVIII production remain unclear. Among organs profiled, only liver and lymph nodes (LNs) show abundant expression of F8 messenger RNA (mRNA). Transcriptomic profiling of subsets of stromal cells, including endothelial cells (ECs) from mouse LNs and other tissues, showed that F8 mRNA is expressed by lymphatic ECs (LECs) but not by capillary ECs (capECs), fibroblastic reticular cells, or hematopoietic cells. Among blood ECs profiled, F8 expression was seen only in fenestrated ECs (liver sinusoidal and renal glomerular ECs) and some high endothelial venules. In contrast, von Willebrand factor mRNA was expressed in capECs but not in LECs; it was coexpressed with F8 mRNA in postcapillary high endothelial venules. Purified LECs and liver sinusoidal ECs but not capECs from LNs secrete active FVIII in culture, and human and mouse lymph contained substantial FVIII: C activity. Our results revealed localized vascular expression of FVIII and von Willebrand factor and identified LECs as a major cellular source of FVIII in extrahepatic tissues.

Angiogenic cytokines are antibody targets during graft-versus-leukemia reactions.

PURPOSE: The graft-versus-leukemia (GVL) reaction is an important example of immune-mediated tumor destruction. A coordinated humoral and cellular response accomplishes leukemia cell killing, but the specific targets remain largely uncharacterized. To learn more about the antigens that elicit antibodies during GVL reactions, we analyzed patients with advanced myelodysplasia (MDS) and acute myelogenous leukemia (AML) who received an autologous, granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting tumor cell vaccine early after allogeneic hematopoietic stem cell transplantation (HSCT). EXPERIMENTAL DESIGN: A combination of tumor-derived cDNA expression library screening, protein microarrays, and antigen-specific ELISAs were used to characterize sera obtained longitudinally from 15 patients with AML/MDS who were vaccinated early after allogeneic HSCT. RESULTS: A broad, therapy-induced antibody response was uncovered, which primarily targeted intracellular proteins that function in growth, transcription/translation, metabolism, and homeostasis. Unexpectedly, antibodies were also elicited against eight secreted angiogenic cytokines that play critical roles in leukemogenesis. Antibodies to the angiogenic cytokines were evident early after therapy, and in some patients manifested a diversification in reactivity over time. Patients that developed antibodies to multiple angiogenic cytokines showed prolonged remission and survival. CONCLUSIONS: These results reveal a potent humoral response during GVL reactions induced with vaccination early after allogeneic HSCT and raise the possibility that antibodies, in conjunction with natural killer cells and T lymphocytes, may contribute to immune-mediated control of myeloid leukemias.

Transcriptional programs of lymphoid tissue capillary and high endothelium reveal control mechanisms for lymphocyte homing.

Lymphocytes are recruited from blood by high-endothelial venules (HEVs). We performed transcriptomic analyses and identified molecular signatures that distinguish HEVs from capillary endothelium and that define tissue-specific HEV specialization. Capillaries expressed gene programs for vascular development. HEV-expressed genes showed enrichment for genes encoding molecules involved in immunological defense and lymphocyte migration. We identify capillary and HEV markers and candidate mechanisms for regulated recruitment of lymphocytes, including a lymph node HEV-selective transmembrane mucin; transcriptional control of functionally specialized carbohydrate ligands for lymphocyte L-selectin; HEV expression of molecules for transendothelial migration; and metabolic programs for lipid mediators of lymphocyte motility and chemotaxis. We also elucidate a carbohydrate-recognition pathway that targets B cells to intestinal lymphoid tissues, defining CD22 as a lectin-homing receptor for mucosal HEVs.

Role of miR-34a as a suppressor of L1CAM in endometrial carcinoma.

L1CAM promotes cell motility, invasion and metastasis formation in various human cancers and can be considered as a driver of tumor progression. Knowledge about genetic processes leading to the presence of L1CAM in cancers is of considerable importance. Experimentally, L1CAM expression can be achieved by various means. Over-expression of the transcription factor SLUG or treatment of cells with TGF-ß1 can induce or augment L1CAM levels in cancer cells. Likewise, hypomethylation of the L1CAM promoter on the X chromosome correlates with L1CAM expression. However, presently no mechanisms that might control transcriptional activity are known. Here we have identified miR-34a as a suppressor of L1CAM. We observed that L1CAM positive endometrial carcinoma (EC) cell lines HEC1B and SPAC1L lost L1CAM protein and mRNA by treatment with demethylating agents or knock-down of the DNA-methyltransferase-1 (DNMT1). Concomitantly, several miRNAs were up-regulated. Using miRNA profiling, luciferase reporter assays and mutagenesis, we identified miR-34a as a putative binder to the L1CAM-3'UTR. Over-expression of miR-34a in HEC1B cells blocked L1CAM expression and inhibited cell migration. In ECC1 cells (wildtype p53) the activation of p53 caused miR-34a up-regulation and loss of L1CAM expression that was miR-34a dependent. We observed an inverse correlation between L1CAM and miR-34a levels in EC cell lines. In primary tumor sections areas expressing high amounts of L1CAM had less miR-34a expression than those with low L1CAM levels. Our data suggest that miR-34a can regulate L1CAM expression by targeting L1CAM mRNA for degradation. These findings shed new light on the complex regulation of L1CAM in human tumors.

EMT-associated up-regulation of L1CAM provides insights into L1CAM-mediated integrin signalling and NF-κB activation.

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-1ß), a process dependent on interaction of L1CAM with integrins. In the present study, we demonstrate that transforming growth factor ß1 (TGF-ß1) 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-1ß and NF-κB activation, which was reversed by depletion of L1CAM, L1CAM-binding membrane cytoskeleton linker protein ezrin, ß1-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 ß1- and α5-integrins, integrin-linked kinase (ILK), FAK and the phosphoinositide 3-kinase (PI3K) subunit p110ß. In summary, these results reveal that during EMT, L1CAM promotes IL-1ß 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.

L1CAM: a major driver for tumor cell invasion and motility.

The L1 cell adhesion molecule (L1CAM) plays a major role in the development of the nervous system and in the malignancy of human tumors. In terms of biological function, L1CAM comes along in two different flavors: (1) a static function as a cell adhesion molecule that acts as a glue between cells; (2) a motility promoting function that drives cell migration during neural development and supports metastasis of human cancers. Important factors that contribute to the switch in the functional mode of L1CAM are: (1) the cleavage from the cell surface by membrane proximal proteolysis and (2) the ability to change binding partners and engage in L1CAM-integrin binding. Recent studies have shown that the cleavage of L1CAM by metalloproteinases and the binding of L1CAM to integrins via its RGD-motif in the sixth Ig-domain activate signaling pathways distinct from the ones elicited by homophilic binding. Here we highlight important features of L1CAM proteolysis and the signaling of L1CAM via integrin engagement. The novel insights into L1CAM downstream signaling and its regulation during tumor progression and epithelial-mesenchymal transition (EMT) will lead to a better understanding of the dualistic role of L1CAM as a cell adhesion and/or motility promoting cell surface molecule.

Combined treatment of L1CAM antibodies and cytostatic drugs improve the therapeutic response of pancreatic and ovarian carcinoma.

The adhesion molecule L1CAM (CD171) accounts for enhanced motility, invasiveness and chemoresistance of tumor cells and represents a novel marker for various tumor entities including pancreatic and ovarian carcinoma. Recently, we showed that L1CAM inhibition increases the apoptotic response of tumor cells towards cytostatic drugs pointing to the potential of L1CAM to serve as a chemosensitizer in anti-cancer therapy. Thus, the present study evaluated the therapeutic potential of combined treatment with L1CAM antibodies and chemotherapeutic drugs in pancreatic and ovarian carcinoma model systems in vivo. Two L1CAM-specific antibodies (L1-14.10 and L1-9.3/2a) exhibiting high binding affinity to the L1CAM expressing pancreatic adenocarcinoma cell line Colo357 and the ovarian carcinoma cell line SKOV3ip were used for treatment. The combined therapy of SCID mice with either L1CAM antibody and gemcitabine and paclitaxel, respectively, reduced the growth of subcutaneously grown Colo357 or SKOV3ip tumors more efficiently than treatment with the cytostatic drug alone or in combination with control IgG. This was accompanied by an increased number of apoptotic tumor cells along with an elevated procaspase-8 expression. Furthermore, a lowered activation of NF-κB along with a reduced expression of VEGF and a diminished number of CD31-positive blood vessels were observed in tumors after combined therapy compared to control treatments, while the infiltration of F4/80-positive macrophages increased. Overall, these data provide new insights into the mechanism of the anti-cancer activity of L1CAM-blocking antibodies in vivo and support the suitability of L1CAM as a target for chemosensitization and of L1CAM-interfering antibodies as an appropriate tool to increase the therapeutic response of pancreatic and ovarian carcinoma.

Myofibroblast-induced tumorigenicity of pancreatic ductal epithelial cells is L1CAM dependent.

Pancreatic ductal adenocarcinoma (PDAC) and chronic pancreatitis, representing one risk factor for PDAC, are characterized by a marked desmoplasia enriched of pancreatic myofibroblasts (PMFs). Thus, PMFs are thought to essentially promote pancreatic tumorigenesis. We recently demonstrated that the adhesion molecule L1CAM is involved in epithelial-mesenchymal transition of PMF-cocultured H6c7 human ductal epithelial cells and that L1CAM is expressed already in ductal structures of chronic pancreatitis with even higher elevation in primary tumors and metastases of PDAC patients. This study aimed at investigating whether PMFs and L1CAM drive malignant transformation of pancreatic ductal epithelial cells by enhancing their tumorigenic potential. Cell culture experiments demonstrated that in the presence of PMFs, H6c7 cells exhibit a profound resistance against death ligand-induced apoptosis. This apoptosis protection was similarly observed in H6c7 cells stably overexpressing L1CAM. Intrapancreatic inoculation of H6c7 cells together with PMFs (H6c7co) resulted in tumor formation in 7/8 and liver metastases in 6/8 severe combined immunodeficiency (SCID) mice, whereas no tumors and metastases were detectable after inoculation of H6c7 cells alone. Likewise, tumor outgrowth and metastases resulted from inoculation of L1CAM-overexpressing H6c7 cells in 5/7 and 3/7 SCID mice, respectively, but not from inoculation of mock-transfected H6c7 cells. Treatment of H6c7co tumor-bearing mice with the L1CAM antibody L1-9.3/2a inhibited tumor formation and liver metastasis in 100 and 50%, respectively, of the treated animals. Overall, these data provide new insights into the mechanisms of how PMFs and L1CAM contribute to malignant transformation of pancreatic ductal epithelial cells in early stages of pancreatic tumorigenesis.

Linking L1CAM-mediated signaling to NF-κB activation.

The cell adhesion molecule L1 (L1CAM) was originally identified as a neural adhesion molecule essential for neurite outgrowth and axon guidance. Many studies have now shown that L1CAM is overexpressed in human carcinomas and associated with poor prognosis. So far, L1CAM-mediated cellular signaling has been largely attributed to an association with growth factor receptors, referred to as L1CAM-'assisted' signaling. New data demonstrate that L1CAM can signal via two additional mechanisms: 'forward' signaling via regulated intramembrane proteolysis and 'reverse' signaling via the activation of the transcription factor nuclear factor (NF)-κB. Taken together, these findings lead to a new understanding of L1CAM downstream signaling that is fundamental for the development of anti-L1CAM antibody-mediated therapeutics in human tumor cells.

Therapeutic antibodies to human L1CAM: functional characterization and application in a mouse model for ovarian carcinoma.

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.

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 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.

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.

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.