Generation and characterisation of laminopathy-specific antibodies

Laminopathien sind eine Gruppe von Krankheiten die hauptsächlich von Mutationen in den A-typ Laminen verursacht werden. Die meisten dieser Mutationen verursachen einen Austausch von nur einer Aminosäure, die meistens autosomal-dominant vererbt werden. Die molekularen Mechanismen von Laminopathien und deren Krankheitsursache, sind aufgrund des Fehlens von spezifischen Forschungswerkzeugen, die das Studieren von den mutanten Proteinen in Patientenzellen erlauben würden, hingegen nur wenig bekannt. Basierend auf dem Ergebnis, dass die im Ogris Labor entwickelten Antikörper gegen Isoformen von Protein Phosphatase 2A (PP2A) Untereinheiten zwischen Isoformen unterscheiden können, die sich nur in einer Aminosäure des entsprechenden Epitops unterscheiden, meinten wir, dass es möglich wäre Antikörper zu entwickeln, die spezifisch das Wildtyp oder die mutanten A-typ Laminproteine, die sich nur in einer Aminosäure unterscheiden, erkennen würden. Solche Antikörper wären für die detailierte Charakterisierung des pathologischen Zustandes und zur Aufklärung der Krankheitsenstehung auf zellulärer Ebene sehr hilfreich. Deshalb hatten wir die Absicht, monoklonale Antikörper spezifisch für autosomal-dominant Emery Dreifuss Muskuläre Dystrophie (EDMD), familiäre partielle Lipodystrophie (FPLD), und dilatierte Kardiomyopathie (DCM) verursachende Punktmutationen in den A-typ Laminen zu entwickeln, die es erlauben würden, zwischen Proteinen, die nur in einer einzigen Aminosäure verschieden sind, zu unterscheiden, um so die speziellen molekularen Eigenschaften des Wildtyp oder des mutanten Proteins zu untersuchen. Um zu solchen Antikörpern zu gelangen wurden drei Ansätze der Antigenpräsentation angewandt. Diese inkludierten den Gebrauch von kurzen Peptiden gekoppelt an das Keyhole limpet hemocyanin (KLH) Protein, rekombinante Proteine bestehend aus der Lamin A/C C-terminalen Ig-fold Domäne fusioniert mit dem Hepatitis B Virus Core Protein (HBcAg), und rekombinante Proteine bestehend aus kurzen Peptiden, mit den gewünschten Lamin A/C Epitopen, fusioniert an HBcAg. Die Entwicklung eines EDMD relevanten punktmutation-spezifischen Antikörpers gegen die Lamin A/C Mutante R453W war mit dem KLH-Peptid Ansatz erfolgreich. Zusätzlich wurde ein Lamin A/C Ig-fold spezifischer monoklonaler Antikörper entwickelt. Bedeutend ist auch, dass Immunisierungen mit dem rekombinanten HBcAg-lamin A/C Ig-fold R482W Fusionsprotein zu einem punktmutations-spezifischen Antikörper führten. Das deutet darauch hin, dass Immunisierungen mit einer ganzen Proteindomäne eine punktmutation-spezifische Immunantwort auslösen können. Bedeutend ist, dass Experimente mit primären EDMD Patientenzellen zeigten, dass der neu entwickelte anti-Lamin A/C R453W Antikörper spezifisch die mutanten Proteine immunpräzipitieren, und die mutanten Proteine in der Immunfluoreszenz und im Immunblotting detektieren konnte. Mit dem anti-lamin A/C R453W Antikörper werden Funktionen der mutanten A-typ Lamine in primären Zellen von EDMD leidenden Patienten untersucht.Laminopathies are a group of diseases caused mainly by mutations in A-type lamins. The vast majority of these mutations cause single amino acid substitutions inherited in an autosomal-dominant way. The molecular mechanisms of laminopathies and their etiology, however, are still poorly understood, mostly because of the lack of specific research tools to study the mutant proteins in patient cells. Based on the finding from the Ogris lab that antibodies against protein phosphatase 2A (PP2A) subunit isoforms can discriminate between isoforms differing only in one amino acid at the respective epitope, we thought that it would be feasible to develop antibodies recognizing specifically the wild-type or the mutant A-type lamin proteins, which are differing only in one amino acid. These antibodies would be extremely helpful for a detailed characterization of the pathological conditions and the elucidation of the disease pathogenesis at the cellular level. Therefore we aimed to develop mouse monoclonal antibodies specific for autosomal-dominant Emery Dreifuss Muscular Dystrophy (EDMD), Familial Partial Lipodystrophy (FPLD), and Dilated Cardiomyopathy (DCM) causing point-mutations in the A-type lamins, which would allow to distinguish between proteins differing in a single amino acid, and thus to study the special molecular features of the wild-type and the mutant proteins, respectively. To obtain such antibodies we made use of three different approaches of antigen presentation. These include the use of short peptides coupled to the keyhole limpet hemocyanin (KLH) protein, recombinant proteins consisting of the lamin A/C C-terminal Ig-fold domain fused to the Hepatitis B virus core protein (HBcAg), and recombinant proteins consisting of short peptides corresponding to the desired lamin A/C epitopes fused to HBcAg. We were successful in developing an EDMD relevant point-mutations specific antibody against the lamin A/C mutant R453W by the KLH-peptide approach. In addition, a lamin A/C Ig-fold specific monoclonal antibody was developed. Importantly, we could also show that a recombinant HBcAg-Lamin A/C Ig-fold R482W fusion protein elicited a humoral immune response resulting in the production of lamin A/C R482W point-mutant specific antibodies, indicating that immunizations with a whole protein domain can elicit a point-mutant specific immune response. Importantly, experiments using primary EDMD patient cells showed that the newly developed anti-lamin A/C R453W antibody could be used to specifically immunoprecipitate the mutant proteins, and to detect the mutant proteins in immunofluorescence and immunoblotting. The anti-lamin A/C R453W antibody will be used to examine the functions of the mutant A-type lamins in primary cells from EDMD patients

TGFβ signaling in myeloid cells promotes lung and liver metastasis through different mechanisms

TGFβ overexpression is commonly detected in cancer patients and correlates with poor prognosis and metastasis. Cancer progression is often associated with an enhanced recruitment of myeloid-derived cells to the tumor microenvironment. Here we show that functional TGFβ-signaling in myeloid cells is required for metastasis to the lungs and the liver. Myeloid-specific deletion of Tgfbr2 resulted in reduced spontaneous lung metastasis, which was associated with a reduction of proinflammatory cytokines in the metastatic microenvironment. Notably, CD8+ T cell depletion in myeloid-specific Tgfbr2-deficient mice rescued lung metastasis. Myeloid-specific Tgfbr2-deficiency resulted in reduced liver metastasis with an almost complete absence of myeloid cells within metastatic foci. On contrary, an accumulation of Tgfβ-responsive myeloid cells was associated with an increased recruitment of monocytes and granulocytes and higher proinflammatory cytokine levels in control mice. Monocytic cells isolated from metastatic livers of Tgfbr2-deficient mice showed increased polarization towards the M1 phenotype, Tnfα and Il-1β expression, reduced levels of M2 markers and reduced production of chemokines responsible for myeloid-cell recruitment. No significant differences in Tgfβ levels were observed at metastatic sites of any model. These data demonstrate that Tgfβ signaling in monocytic myeloid cells suppresses CD8+ T cell activity during lung metastasis, while these cells actively contribute to tumor growth during liver metastasis. Thus, myeloid cells modulate metastasis through different mechanisms in a tissue-specific manner

Fos regulates macrophage infiltration against surrounding tissue resistance by a cortical actin-based mechanism in Drosophila

The infiltration of immune cells into tissues underlies the establishment of tissue-resident macrophages and responses to infections and tumors. Yet the mechanisms immune cells utilize to negotiate tissue barriers in living organisms are not well understood, and a role for cortical actin has not been examined. Here, we find that the tissue invasion of Drosophila macrophages, also known as plasmatocytes or hemocytes, utilizes enhanced cortical F-actin levels stimulated by the Drosophila member of the fos proto oncogene transcription factor family (Dfos, Kayak). RNA sequencing analysis and live imaging show that Dfos enhances F-actin levels around the entire macrophage surface by increasing mRNA levels of the membrane spanning molecular scaffold tetraspanin TM4SF, and the actin cross-linking filamin Cheerio, which are themselves required for invasion. Both the filamin and the tetraspanin enhance the cortical activity of Rho1 and the formin Diaphanous and thus the assembly of cortical actin, which is a critical function since expressing a dominant active form of Diaphanous can rescue the Dfos macrophage invasion defect. In vivo imaging shows that Dfos enhances the efficiency of the initial phases of macrophage tissue entry. Genetic evidence argues that this Dfos-induced program in macrophages counteracts the constraint produced by the tension of surrounding tissues and buffers the properties of the macrophage nucleus from affecting tissue entry. We thus identify strengthening the cortical actin cytoskeleton through Dfos as a key process allowing efficient forward movement of an immune cell into surrounding tissues

The Solute Carrier MFSD1 Decreases the Activation Status of β1 Integrin and Thus Tumor Metastasis

Solute carriers are increasingly recognized as participating in a plethora of pathologies, including cancer. We describe here the involvement of the orphan solute carrier Major Facilitator Superfamily Domain-containing protein 1 (MFSD1) in the regulation of tumor cell migration. Loss of MFSD1 enabled higher levels of metastasis in experimental and spontaneous metastasis mouse models. We identified an increased migratory potential in MFSD1-/- tumor cells which was mediated by increased focal adhesion turnover, reduced stability of mature inactive β1 integrin, and the resulting increased integrin activation index. We show that MFSD1 promoted recycling to the cell surface of endocytosed inactive β1 integrin and thereby protected β1 integrin from proteolytic degradation; this led to dampening of the integrin activation index. Furthermore, downregulation of MFSD1 expression was observed during the early steps of tumorigenesis, and higher MFSD1 expression levels correlate with a better cancer patient prognosis. In sum, we describe a requirement for endolysosomal MFSD1 in efficient β1 integrin recycling to suppress tumor cell dissemination

Single cell polarity in liquid phase facilitates tumour metastasis

Dynamic polarisation of tumour cells is essential for metastasis. While the role of polarisation during dedifferentiation and migration is well established, polarisation of metastasising tumour cells during phases of detachment has not been investigated. Here we identify and characterise a type of polarisation maintained by single cells in liquid phase termed single-cell (sc) polarity and investigate its role during metastasis. We demonstrate that sc polarity is an inherent feature of cells from different tumour entities that is observed in circulating tumour cells in patients. Functionally, we propose that the sc pole is directly involved in early attachment, thereby affecting adhesion, transmigration and metastasis. In vivo, the metastatic capacity of cell lines correlates with the extent of sc polarisation. By manipulating sc polarity regulators and by generic depolarisation, we show that sc polarity prior to migration affects transmigration and metastasis in vitro and in vivo

A conserved major facilitator superfamily member orchestrates a subset of O-glycosylation to aid macrophage tissue invasion

Aberrant display of the truncated core1 O-glycan T-antigen is a common feature of human cancer cells that correlates with metastasis. Here we show that T-antigen in Drosophila melanogaster macrophages is involved in their developmentally programmed tissue invasion. Higher macrophage T-antigen levels require an atypical major facilitator superfamily (MFS) member that we named Minerva which enables macrophage dissemination and invasion. We characterize for the first time the T and Tn glycoform O-glycoproteome of the Drosophila melanogaster embryo, and determine that Minerva increases the presence of T-antigen on proteins in pathways previously linked to cancer, most strongly on the sulfhydryl oxidase Qsox1 which we show is required for macrophage tissue entry. Minerva’s vertebrate ortholog, MFSD1, rescues the minerva mutant’s migration and T-antigen glycosylation defects. We thus identify a key conserved regulator that orchestrates O-glycosylation on a protein subset to activate a program governing migration steps important for both development and cancer metastasis

Endothelial CCR2 Promotes Pulmonary Metastasis

Nine out of ten cancer patients die due to metastasis. The generation of them is dependent on several factors, including the recruitment of inflammatory monocytes and activation of endothelial cells at the pre-metastatic niche. It is at these sites where cancer cell seeding of the targeted secondary tissue is taking place. Inflammatory chemokines, especially the chemokine ligand CCL2 and its receptor CCR2, are regulating several aspects of the metastatic cascade, including monocyte recruitment to the metastatic niche and induction of vascular permeability enabling cancer cell trans-endothelial migration. We aimed to target the CCL2-CCR2 signaling axis at the pre-metastatic niche by pharmacological inhibition and a genetic endothelial specific CCR2 knockout (CCR2ecKO) mouse model. A mutant signaling deficient dnCCL2-HSA chimera strongly inhibited cancer cell trans-endothelial migration as well as cancer cell seeding to the lung. As a consequence the metastatic burden was strongly decreased in dnCCL2-HSA treated mice. The anti-metastatic effect of dnCCL2-HSA was dependent on its inhibition of cancer cell induced vascular permeability, while recruitment of inflammatory monocytes to the pre-metastatic niche was not impaired. dnCCL2-HSA was found to be present in close proximity to cancer cells in the lung vasculature, indicating that local inhibition of CCR2 signaling is sufficient to prevent metastasis initiation. Furthermore, targeting of cancer cell activated endothelial cells by VCAM-1 binding peptide equipped liposomes loaded with a CCR2 antagonist confirmed that inhibition of CCR2 at sites of activated endothelium is sufficient to prevent the generation of metastases. The spatial inhibition of CCR2 using these liposomes interfered with induction of vascular permeability, but spared any detectable effects on inflammatory monocytes. The endothelial specific CCR2 knockout (CCR2ecKO) mouse model confirmed findings from both inhibitor approaches. Spontaneous lung metastasis was strongly reduced in CCR2ecKO mice compared to control littermates. Leukocyte recruitment to the primary tumor, the pre-metastatic niche, and the metastatic environment however, was not affected due to the endothelial CCR2 knockout.Stimulation of endothelial cells with CCL2 caused phosphorylation of MLC2, which is required for endothelial cell retraction. Overall we could show that endothelial CCR2 is the gatekeeper for cancer cell trans-endothelial migration and thus represents the final check-point for metastasis initiation of colon and lung cancer cells. Targeting endothelial CCR2 at pre-metastatic sites represents a promising treatment strategy to prevent cancer cell seeding and thus metastatic spread to the lung

TGFβ Signaling in Myeloid Cells Promotes Lung and Liver Metastasis Through Different Mechanisms

TGFβ overexpression is commonly detected in cancer patients and correlates with poor prognosis and metastasis. Cancer progression is often associated with an enhanced recruitment of myeloid-derived cells to the tumor microenvironment. Here we show that functional TGFβ-signaling in myeloid cells is required for metastasis to the lungs and the liver. Myeloid-specific deletion of Tgfbr2 resulted in reduced spontaneous lung metastasis, which was associated with a reduction of proinflammatory cytokines in the metastatic microenvironment. Notably, CD8$^{+}$ T cell depletion in myeloid-specific Tgfbr2-deficient mice rescued lung metastasis. Myeloid-specific Tgfbr2-deficiency resulted in reduced liver metastasis with an almost complete absence of myeloid cells within metastatic foci. On contrary, an accumulation of Tgfβ-responsive myeloid cells was associated with an increased recruitment of monocytes and granulocytes and higher proinflammatory cytokine levels in control mice. Monocytic cells isolated from metastatic livers of Tgfbr2-deficient mice showed increased polarization towards the M1 phenotype, Tnfα and Il-1β expression, reduced levels of M2 markers and reduced production of chemokines responsible for myeloid-cell recruitment. No significant differences in Tgfβ levels were observed at metastatic sites of any model. These data demonstrate that Tgfβ signaling in monocytic myeloid cells suppresses CD8$^{+}$ T cell activity during lung metastasis, while these cells actively contribute to tumor growth during liver metastasis. Thus, myeloid cells modulate metastasis through different mechanisms in a tissue-specific manner

The role of VLA-4 binding for experimental melanoma metastasis and its inhibition by heparin

Introduction: Heparin is known to efficiently attenuate metastasis in various tumour models by different mechanisms including inhibition of tumour cell contacts with soluble and cellular components such as inhibition of heparanase or P- and L-selectin. We recently showed that heparin efficiently binds to VLA-4 integrin in melanoma cells in vitro. Here we describe VLA-4 integrin as a mediator of melanoma metastasis that is inhibited by the low molecular weight heparin (LMWH) Tinzaparin. Materials and Methods: sh-RNA-mediated knock-down of VLA-4 integrin in B16F10 murine melanoma cells (B16F10-VLA-4kd) was performed and cell binding characteristics were investigated in vitro. Experimental metastasis of B16F10-VLA-4kd and B16F10 cells and interference by Tinzaparin were analysed in mice. Results: VLA-4 knock-down of B16F10 cells resulted in loss of VCAM-1 binding, but preserved the capacity to bind platelets through P-selectin. The observed reduced metastasis of B16F10-VLA-4kd cells confirmed the role of VLA-4 in this process. However, loss of melanoma VLA-4 function hardly further affected reduction of metastasis in P-selectin deficient mice. Tinzaparin treatment of mice injected with B16F10 and B16F10-VLA-4kd cells significantly reduced metastasis suggesting its potential to block both P- and L-selectin and VLA-4 in vivo. The use of N-acetylated heparin, which has no VLA-4 binding activity but blocks P- and L-selectin was less efficient than Tinzaparin in mice injected with B16F10 cells and B16F10-VLA-4kd cells. Conclusion: These findings provide evidence that heparin inhibits experimental melanoma metastasis primarily by blocking VLA-4 and P-selectin

Monoclonal antibodies specific for disease-associated point-mutants: lamin A/C R453W and R482W.

Disease-linked missense mutations can alter a protein's function with fatal consequences for the affected individual. How a single amino acid substitution in a protein affects its properties, is difficult to study in the context of the cellular proteome, because mutant proteins can often not be traced in cells due to the lack of mutation-specific detection tools. Antibodies, however, with their exquisite epitope specificity permit the detection of single amino acid substitutions but are not available for the vast majority of disease-causing mutant proteins. One of the most frequently missense-mutated human genes is the LMNA gene coding for A-type lamins. Mutations in LMNA cause phenotypically heterogenous, mostly autosomal-dominant inherited diseases, termed laminopathies. The molecular mechanisms underlying the phenotypic heterogeneity of laminopathies, however, are not well understood. Hence, the goal of this study was the development of monoclonal antibodies specific for disease-linked point-mutant A-type lamins.Using two different approaches of antigen presentation, namely KLH-coupled peptides and the display of a complete protein domain fused to the Hepatitis B virus capsid protein, we developed monoclonal antibodies against two disease-associated lamin A/C mutants. Both antibodies display exquisite specificity for the respective mutant proteins. We show that with the help of these novel antibodies it is now possible for the first time to study specifically the properties of the mutant proteins in primary patient cells in the background of wild-type protein.We report here the development of two point-mutant specific antibodies against A-type lamins. While synthetic peptides may be the prime choice of antigen, our results show that a given target sequence may have to be presented in alternative ways to ensure the induction of a mutant-specific immune response. Point-mutant specific antibodies will represent valuable tools for basic and clinical research on a number of hereditary as well as acquired diseases caused by dominant missense mutations