Glucocorticoid activation by HSD11B1 limits T cell-driven interferon signaling and response to PD-1 blockade in melanoma.

BACKGROUND: Immune responses against tumors are subject to negative feedback regulation. Immune checkpoint inhibitors (ICIs) blocking Programmed cell death protein 1 (PD-1), a receptor expressed on T cells, or its ligand PD-L1 have significantly improved the treatment of cancer, in particular malignant melanoma. Nevertheless, responses and durability are variables, suggesting that additional critical negative feedback mechanisms exist and need to be targeted to improve therapeutic efficacy. METHODS: We used different syngeneic melanoma mouse models and performed PD-1 blockade to identify novel mechanisms of negative immune regulation. Genetic gain-of-function and loss-of-function approaches as well as small molecule inhibitor applications were used for target validation in our melanoma models. We analyzed mouse melanoma tissues from treated and untreated mice by RNA-seq, immunofluorescence and flow cytometry to detect changes in pathway activities and immune cell composition of the tumor microenvironment. We analyzed tissue sections of patients with melanoma by immunohistochemistry as well as publicly available single-cell RNA-seq data and correlated target expression with clinical responses to ICIs. RESULTS: Here, we identified 11-beta-hydroxysteroid dehydrogenase-1 (HSD11B1), an enzyme that converts inert glucocorticoids into active forms in tissues, as negative feedback mechanism in response to T cell immunotherapies. Glucocorticoids are potent suppressors of immune responses. HSD11B1 was expressed in different cellular compartments of melanomas, most notably myeloid cells but also T cells and melanoma cells. Enforced expression of HSD11B1 in mouse melanomas limited the efficacy of PD-1 blockade, whereas small molecule HSD11B1 inhibitors improved responses in a CD8+ T cell-dependent manner. Mechanistically, HSD11B1 inhibition in combination with PD-1 blockade augmented the production of interferon-γ by T cells. Interferon pathway activation correlated with sensitivity to PD-1 blockade linked to anti-proliferative effects on melanoma cells. Furthermore, high levels of HSD11B1, predominantly expressed by tumor-associated macrophages, were associated with poor responses to ICI therapy in two independent cohorts of patients with advanced melanomas analyzed by different methods (scRNA-seq, immunohistochemistry). CONCLUSION: As HSD11B1 inhibitors are in the focus of drug development for metabolic diseases, our data suggest a drug repurposing strategy combining HSD11B1 inhibitors with ICIs to improve melanoma immunotherapy. Furthermore, our work also delineated potential caveats emphasizing the need for careful patient stratification.

Targeting early stages of cardiotoxicity from anti-PD1 immune checkpoint inhibitor therapy.

AIMS: Cardiac immune-related adverse events (irAEs) from immune checkpoint inhibition (ICI) targeting programmed death 1 (PD1) are of growing concern. Once cardiac irAEs become clinically manifest, fatality rates are high. Cardio-oncology aims to prevent detrimental effects before manifestation of severe complications by targeting early pathological changes. We therefore aimed to investigate early consequences of PD1 inhibition for cardiac integrity to prevent the development of overt cardiac disease. METHODS AND RESULTS: We investigated cardiac-specific consequences from anti-PD1 therapy in a combined biochemical and in vivo phenotyping approach. Mouse hearts showed broad expression of the ligand PDL1 on cardiac endothelial cells as a main mediator of immune-crosstalk. Using a novel melanoma mouse model, we assessed that anti-PD1 therapy promoted myocardial infiltration with CD4+ and CD8+ T cells, the latter being markedly activated. Left ventricular (LV) function was impaired during pharmacological stress, as shown by pressure-volume catheterization. This was associated with a dysregulated myocardial metabolism, including the proteome and the lipidome. Analogous to the experimental approach, in patients with metastatic melanoma (n = 7) receiving anti-PD1 therapy, LV function in response to stress was impaired under therapy. Finally, we identified that blockade of tumour necrosis factor alpha (TNFα) preserved LV function without attenuating the anti-cancer efficacy of anti-PD1 therapy. CONCLUSIONS: Anti-PD1 therapy induces a disruption of cardiac immune homeostasis leading to early impairment of myocardial functional integrity, with potential prognostic effects on the growing number of treated patients. Blockade of TNFα may serve as an approach to prevent the manifestation of ICI-related cardiotoxicity.

Allosteric Activation of GDP-Bound Ras Isoforms by Bisphenol Derivative Plasticisers.

The protein family of small GTPases controls cellular processes by acting as a binary switch between an active and an inactive state. The most prominent family members are H-Ras, N-Ras, and K-Ras isoforms, which are highly related and frequently mutated in cancer. Bisphenols are widespread in modern life because of their industrial application as plasticisers. Bisphenol A (BPA) is the best-known member and has gained significant scientific as well as public attention as an endocrine disrupting chemical, a fact that eventually led to its replacement. However, compounds used to replace BPA still contain the molecular scaffold of bisphenols. BPA, BPAF, BPB, BPE, BPF, and an amine-substituted BPAF-derivate all interact with all GDP-bound Ras-Isoforms through binding to a common site on these proteins. NMR-, SOScat-, and GDI- assay-based data revealed a new bisphenol-induced, allosterically activated GDP-bound Ras conformation that define these plasticisers as Ras allosteric agonists.

Targeting of Cell Surface Proteolysis of Collagen XVII Impedes Squamous Cell Carcinoma Progression.

Squamous cell carcinoma (SCC) is one of the most common skin cancers and causes significant morbidity. Although the expression of the epithelial adhesion molecule collagen XVII (ColXVII) has been linked to SCC invasion, only little is known about its mechanistic contribution. Here, we demonstrate that ColXVII expression is essential for SCC cell proliferation and motility. Moreover, it revealed that particularly the post-translational modification of ColXVII by ectodomain shedding is the major driver of SCC progression, because ectodomain-selective immunostaining was mainly localized at the invasive front of human cutaneous SCCs, and exclusive expression of a non-sheddable ColXVII mutant in SCC-25 cells inhibits their matrix-independent growth and invasiveness. This cell surface proteolysis, which is strongly elevated during SCC invasion and metastasis, releases soluble ectodomains and membrane-anchored endodomains. Both released ColXVII domains play distinct roles in tumor progression: the endodomain induces proliferation and survival, whereas the ectodomain accelerates invasiveness. Furthermore, specific blockage of shedding by monoclonal ColXVII antibodies repressed matrix-independent growth and invasion of SCC cells in organotypic co-cultures. Thus, selective inhibition of ColXVII shedding may offer a promising therapeutic strategy to prevent SCC progression.

Transforming of the Tumor Microenvironment: Implications for TGF-ß Inhibition in the Context of Immune-Checkpoint Therapy.

Significant breakthroughs have been achieved in the fields of oncogenic signaling inhibition and particularly immune-checkpoint blockade has triggered substantial enthusiasm during the last decade. Antibody-mediated blockade of negative immune-checkpoint molecules (e.g., PD-1/PD-L1, CTLA-4) has been shown to achieve profound responses in several of solid cancers. Unfortunately, these responses only occur in a subset of patients or, after initial therapy response, these tumors eventually relapse. Thus, elucidating the determinants of intrinsic or therapy-induced resistance is the key to improve outcomes and developing new treatment strategies. Several cytokines and growth factors are involved in the tight regulation of either antitumor immunity or immunosuppressive tumor-promoting inflammation within the tumor microenvironment (TME), of which transforming growth factor beta (TGF-ß) is of particular importance. This review will therefore summarize the recent progress that has been made in the understanding of how TGF-ß blockade may have the capacity to enhance efficacy of immune-checkpoint therapy which presents a rational strategy to sustain the antitumor inflammatory response to improve response rates in tumor patients. Finally, I will conclude with a comprehensive summary of clinical trials in which TGF-ß blockade revealed therapeutic benefit for patients by counteracting tumor relapses.

Significant Role of Collagen XVII And Integrin ß4 in Migration and Invasion of The Less Aggressive Squamous Cell Carcinoma Cells.

Collagen XVII and integrin α6ß4 have well-established roles as epithelial adhesion molecules. Their binding partner laminin 332 as well as integrin α6ß4 are largely recognized to promote invasion and metastasis in various cancers, and collagen XVII is essential for the survival of colon and lung cancer stem cells. We have studied the expression of laminin γ2, collagen XVII and integrin ß4 in tissue microarray samples of squamous cell carcinoma (SCC) and its precursors, actinic keratosis and Bowen's disease. The expression of laminin γ2 was highest in SCC samples, whereas the expression of collagen XVII and integrin ß4 varied greatly in SCC and its precursors. Collagen XVII and integrin ß4 were also expressed in SCC cell lines. Virus-mediated RNAi knockdown of collagen XVII and integrin ß4 reduced the migration of less aggressive SCC-25 cells in horizontal scratch wound healing assay. Additionally, in a 3D organotypic myoma invasion assay the loss of collagen XVII or integrin ß4 suppressed equally the migration and invasion of SCC-25 cells whereas there was no effect on the most aggressive HSC-3 cells. Variable expression patterns and results in migration and invasion assays suggest that collagen XVII and integrin ß4 contribute to SCC tumorigenesis.

Tension in Cancer.

Integrins represent a large family of cell receptors that mediate adhesion to the extracellular matrix (ECM), thereby modulating a variety of cellular functions that are required for proliferation, migration, malignant conversion and invasiveness. During tumorigenesis the conversion of a tumor cell from sessile, stationary phenotype to an invasive phenotype requires the ability of tumor cells to interact with their environment in order to transduce signals from the ECM into the cells. Hence, there is increasing evidence that changes in the composition, topography and tension of tumor matrix can be sensed by integrin receptors, leading to the regulation of intracellular signalling events which subsequently help to fuel cancer progression. The fact that intracellular signals perceived from integrin ligand binding impact on almost all steps of tumor progression, including tumor cell proliferation, survival, metastatic dissemination and colonization of a metastatic niche, renders integrins as ideal candidates for the development of therapeutic agents. In this review we summarize the role of integrins in cancer with the special focus on cancer therapies and the recent progress that has been made in the understanding of "integrin-induced tension in cancer". Finally, we conclude with clinical evidence for the role of integrin-mediated mechanotransduction in the development of therapy-resistant tumors.

Skin Barrier Defects Caused by Keratinocyte-Specific Deletion of ADAM17 or EGFR Are Based on Highly Similar Proteome and Degradome Alterations.

Keratinocyte-specific deletion of ADAM17 in mice impairs terminal differentiation of keratinocytes leading to severe epidermal barrier defects. Mice deficient for ADAM17 in keratinocytes phenocopy mice with a keratinocyte-specific deletion of epidermal growth factor receptor (EGFR), which highlights the role of ADAM17 as a "ligand sheddase" of EGFR ligands. In this study, we aim for the first proteomic/degradomic approach to characterize the disruption of the ADAM17-EGFR signaling axis and its consequences for epidermal barrier formation. Proteomic profiling of the epidermal proteome of mice deficient for either ADAM17 or EGFR in keratinocytes at postnatal days 3 and 10 revealed highly similar protein alterations for ADAM17 and EGFR deficiency. These include massive proteome alterations of structural and regulatory components important for barrier formation such as transglutaminases, involucrin, filaggrin, and filaggrin-2. Cleavage site analysis using terminal amine isotopic labeling of substrates revealed increased proteolytic processing of S100 fused-type proteins including filaggrin-2. Alterations in proteolytic processing are supported by altered abundance of numerous proteases upon keratinocyte-specific Adam17 or Egfr deletion, among them kallikreins, cathepsins, and their inhibitors. This study highlights the essential role of proteolytic processing for maintenance of a functional epidermal barrier. Furthermore, it suggests that most defects in formation of the postnatal epidermal barrier upon keratinocyte-specific ADAM17 deletion are mediated via EGFR.

Collagen XVII Shedding Suppresses Re-Epithelialization by Directing Keratinocyte Migration and Dampening mTOR Signaling.

Transmembrane collagen XVII is traditionally viewed as an important hemidesmosomal attachment component that promotes stable dermal-epidermal adhesion in the skin. However, its expression is highly elevated at the leading edges of cutaneous wounds or invasive carcinomas, suggesting alternative functions in cell migration. The collagenous ectodomain of collagen XVII is constitutively shed from the cell surface by a disintegrin and metalloproteinases, and this shedding is strongly induced during wound healing. Recently, we investigated the physiological relevance of collagen XVII shedding by generating knock-in mice, which exclusively express a functional non-sheddable collagen XVII mutant. Prevention of ectodomain shedding in these mice caused no spontaneous phenotype in resting skin, but accelerated re-epithelialization on skin wounding. Here, we investigated the mechanistic function of shedding during wound healing. Using the non-shedding collagen XVII mice as a model, we uncovered ectodomain shedding as a highly dynamic modulator of in vivo proliferation and motility of activated keratinocytes through tight coordination of α6ß4 integrin-laminin-332 interactions and dampening of mechanistic target of rapamycin signaling at the wound edges. Thus, our studies identify ectodomain shedding of collagen XVII as an interactive platform that translates shedding into a signal for directed cell growth and motility during skin regeneration.

Injury-Driven Stiffening of the Dermis Expedites Skin Carcinoma Progression.

Recessive dystrophic epidermolysis bullosa (RDEB) is a genetic skin fragility disorder characterized by injury-driven blister formation, progressive soft-tissue fibrosis, and a highly elevated risk of early-onset aggressive cutaneous squamous cell carcinoma (cSCC). However, the mechanisms underlying the unusually rapid progression of RDEB to cSCC are unknown. In this study, we investigated the contribution of injury-induced skin alterations to cSCC development by using a genetic model of RDEB and organotypic skin cultures. Analysis of RDEB patient samples suggested that premalignant changes to the dermal microenvironment drive tumor progression, which led us to subject a collagen VII hypomorphic mouse model of RDEB to chemical carcinogenesis. Carcinogen-treated RDEB mice developed invasive tumors phenocopying human RDEB-cSCC, whereas wild-type mice formed papillomas, indicating that the aggressiveness of RDEB-cSCC is mutation-independent. The inherent structural instability of the RDEB dermis, combined with repeated injury, increased the bioavailability of TGFß, which promoted extracellular matrix production, cross-linking, thickening of dermal fibrils, and tissue stiffening. The biophysically altered dermis increased myofibroblast activity and integrin ß1/pFAK/pAKT mechanosignaling in tumor cells, further demonstrating that cSCC progression is governed by pre-existing injury-driven changes in the RDEB tissue microenvironment. Treatment of three-dimensional organotypic RDEB skin cultures with inhibitors of TGFß signaling, lysyl oxidase, or integrin ß1-mediated mechanosignaling reduced or bypassed tissue stiffness and limited tumor cell invasion. Collectively, these findings provide a new mechanism by which RDEB tissue becomes malignant and offer new druggable therapeutic targets to prevent cSCC onset.

CEACAM1-4L Promotes Anchorage-Independent Growth in Melanoma.

Widespread metastasis is the leading course of death in many types of cancer, including malignant melanoma. The process of metastasis can be divided into a number of complex cell biological events, collectively termed the "invasion-metastasis cascade." Previous reports have characterized the capability of anchorage-independent growth of cancer cells in vitro as a key characteristic of highly aggressive tumor cells, particularly with respect to metastatic potential. Biological heterogeneity as well as drastic alterations in cell adhesion of disseminated cancer cells support escape mechanisms for metastases to overcome conventional therapies. Here, we show that exclusively the carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) splice variant CEACAM1-4L supports an anchorage-independent signature in malignant melanoma. These results highlight important variant-specific modulatory functions of CEACAM1 for metastatic spread in patients suffering malignant melanoma.

MITF is a critical regulator of the carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) in malignant melanoma.

The multifunctional Ig-like carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is neo-expressed in the majority of malignant melanoma lesions. CEACAM1 acts as a driver of tumor cell invasion, and its expression correlates with poor patient prognosis. Despite its importance in melanoma progression, how CEACAM1 expression is regulated is largely unknown. Here, we show that CEACAM1 expression in melanoma cell lines and melanoma tissue strongly correlates with that of the microphthalmia-associated transcription factor (MITF), a key regulator of melanoma proliferation and invasiveness. MITF is revealed as a direct and positive regulator for CEACAM1 expression via binding to an M-box motif located in the CEACAM1 promoter. Taken together, our study provides novel insights into the regulation of CEACAM1 expression and suggests an MITF-CEACAM1 axis as a potential determinant of melanoma progression.

Collagen XVII expression correlates with the invasion and metastasis of colorectal cancer.

Collagen XVII has a well-established role as an adhesion molecule and a cell surface receptor located in the type I hemidesmosome of stratified epithelia. Its ectodomain is constitutively shed from the cell surface and suggested to regulate the adhesion, migration, and signaling of cutaneous epithelial cells. Collagen XVII was not previously thought to be expressed by colon epithelial cells. Immunohistochemical analysis of tissue microarray samples of 141 cases of colorectal carcinoma showed that collagen XVII is expressed in normal human colonic mucosa and colorectal carcinoma. In colorectal carcinoma, increased collagen XVII expression was significantly associated with higher TNM stage. It also correlated with infiltrative growth pattern and tumor budding as well as lymph node and distant metastasis. Increased collagen XVII expression was associated with decreased disease-free and cancer-specific survival. Immunofluorescence staining of collagen XVII and its well-known binding partner laminin γ2 chain demonstrated a partial colocalization in normal and tumor tissue. In vitro, the overexpression of murine collagen XVII promoted the invasion of CaCo-2 colon carcinoma cells through Matrigel (BD Biosciences; Bedford, MA). To conclude, this study reports for the first time the expression of collagen XVII in colon epithelium and the association of increased collagen XVII immunoexpression with poor outcome in colorectal carcinoma.

Deletion of the major bullous pemphigoid epitope region of collagen XVII induces blistering, autoimmunization, and itching in mice.

Bullous pemphigoid (BP) is the most common autoimmune subepidermal blistering skin disease with a characteristic of pruritus and blistering. BP patients carry inflammation-triggering autoantibodies against the collagen XVII (ColXVII, also known as BP180) juxtamembraneous extracellular noncollagenous 16A (NC16A) domain involved in ectodomain shedding. Deletion of the corresponding NC14A region in a genetically modified mouse model (ΔNC14A) decreased the amount of ColXVII in skin, but it did not prevent ectodomain shedding. Newborn ΔNC14A mice had no macroscopic phenotypic changes. However, subepidermal microblisters, rudimentary hemidesmosomes, and loose basement membrane zone were observed by microscopy. ΔNC14A mice grow normally, but at around 3 months of age they start to scratch themselves and develop crusted erosions. Furthermore, perilesional eosinophilic infiltrations in the skin, eosinophilia, and elevated serum IgE levels are detected. Despite the removal of the major BP epitope region, ΔNC14A mice developed IgG and IgA autoantibodies with subepidermal reactivity, indicating autoimmunization against a dermo-epidermal junction component. Moreover, IgG autoantibodies recognized a 180-kDa keratinocyte protein, which was sensitive to collagenase digestion. We show here that ΔNC14A mice provide a highly reproducible BP-related mouse model with spontaneous breakage of self-tolerance and development of autoantibodies.

Transmembrane collagen XVII modulates integrin dependent keratinocyte migration via PI3K/Rac1 signaling.

The hemidesmosomal transmembrane component collagen XVII (ColXVII) plays an important role in the anchorage of the epidermis to the underlying basement membrane. However, this adhesion protein seems to be also involved in the regulation of keratinocyte migration, since its expression in these cells is strongly elevated during reepithelialization of acute wounds and in the invasive front of squamous cell carcinoma, while its absence in ColXVII-deficient keratinocytes leads to altered cell motility. Using a genetic model of murine Col17a1⁻/⁻ keratinocytes we elucidated ColXVII mediated signaling pathways in cell adhesion and migration. Col17a1⁻/⁻ keratinocytes exhibited increased spreading on laminin 332 and accelerated, but less directed cell motility. These effects were accompanied by increased expression of the integrin subunits ß4 and ß1. The migratory phenotype, as evidenced by formation of multiple unstable lamellipodia, was associated with enhanced phosphoinositide 3-kinase (PI3K) activity. Dissection of the signaling pathway uncovered enhanced phosphorylation of the ß4 integrin subunit and the focal adhesion kinase (FAK) as activators of PI3K. This resulted in elevated Rac1 activity as a downstream consequence. These results provide mechanistic evidence that ColXVII coordinates keratinocyte adhesion and directed motility by interfering integrin dependent PI3K activation and by stabilizing lamellipodia at the leading edge of reepithelializing wounds and in invasive squamous cell carcinoma.

Involvement of the ubiquitin-proteasome system in the stabilization of cell-cell contacts in human keratinocytes.

Desmosomes are highly organized intercellular junctions composed of a number of interacting proteins that provide mechanical integrity to epithelial tissues. Mutations in genes encoding desmosomal proteins, including desmoplakin (DP), are associated with human hereditary diseases affecting skin integrity. The detailed mechanism of desmosome assembly remains, despite many efforts, incompletely understood. Recently, the ubiquitin-proteasome system (UPS) has been suggested to be an important regulatory system for the proper intracellular trafficking of proteins. Here, we provide evidence for a calcium-independent, but UPS-dependent, stabilization of cell-cell contacts in human keratinocytes, which might be mediated by the maintenance of DP at desmosomes.

Epidermal ADAM17 maintains the skin barrier by regulating EGFR ligand-dependent terminal keratinocyte differentiation.

ADAM17 (a disintegrin and metalloproteinase 17) is ubiquitously expressed and cleaves membrane proteins, such as epidermal growth factor receptor (EGFR) ligands, l-selectin, and TNF, from the cell surface, thus regulating responses to tissue injury and inflammation. However, little is currently known about its role in skin homeostasis. We show that mice lacking ADAM17 in keratinocytes (A17(ΔKC)) have a normal epidermal barrier and skin architecture at birth but develop pronounced defects in epidermal barrier integrity soon after birth and develop chronic dermatitis as adults. The dysregulated expression of epidermal differentiation proteins becomes evident 2 d after birth, followed by reduced transglutaminase (TGM) activity, transepidermal water loss, up-regulation of the proinflammatory cytokine IL-36α, and inflammatory immune cell infiltration. Activation of the EGFR was strongly reduced in A17(ΔKC) skin, and topical treatment of A17(ΔKC) mice with recombinant TGF-α significantly improved TGM activity and decreased skin inflammation. Finally, we show that mice lacking the EGFR in keratinocytes (Egfr(ΔKC)) closely resembled A17(ΔKC) mice. Collectively, these results identify a previously unappreciated critical role of the ADAM17-EGFR signaling axis in maintaining the homeostasis of the postnatal epidermal barrier and suggest that this pathway could represent a good target for treatment of epidermal barrier defects.

The ubiquitin ligase CHIP/STUB1 targets mutant keratins for degradation.

Keratin (K) intermediate filament proteins form cytoskeletal scaffolds in epithelia, the disruption of which leads to a large number of human disorders. KRT5 or KRT14 mutations cause epidermolysis bullosa simplex (EBS). The considerable intra- and interfamilial variability in EBS suggests modifying loci, most of which are unknown. In many human disorders, chaperones and the ubiquitin-proteasome system have been found to modify disease severity, thereby providing novel therapy targets. Here, we demonstrate upregulation of stress-induced Hsp70 and Hsp90 in two EBS models, namely, in neonatal K5(-/-) mice and upon proteasome inhibition in cells that stably express the disease-causing mutation K14-p.Arg125Cys, both harboring keratin aggregates. Furthermore, proteasome inhibition caused nuclear translocation of pHSF-1 and an increase in K14-p.Arg125Cys-positive aggregates in cells. Overexpression of the chaperone-associated ubiquitin ligase CHIP/STUB1 strongly reduced keratin aggregates through increased degradation of mutant K14. Using CHIP-p.Met1_Ala142del (DeltaTPR-CHIP), we demonstrated the involvement of Hsc70 and Hsp70 in mutant keratin degradation. Our data uncover common principles between EBS and other protein misfolding disorders, revealing that aggregation-prone keratins are targeted by components of the chaperone machinery. Thus, modulation of the chaperone machinery using small molecules may represent a novel therapeutic strategy for dominant EBS, allowing reformation of an intact keratin cytoskeleton.

Role of ADAM-9 disintegrin-cysteine-rich domains in human keratinocyte migration.

ADAM-9 belongs to a family of transmembrane, disintegrin-containing metalloproteinases involved in protein ectodomain shedding and cell-cell and cell-matrix interactions. The aim of this study was to analyze the expression of ADAM-9 in skin and to assess the role of this proteolytic/adhesive protein in skin physiology. In normal skin, ADAM-9 expression was detected in both the epidermis and dermis and in vitro in keratinocytes and fibroblasts. Here we report that ADAM-9 functions as a cell adhesion molecule via its disintegrin-cysteine-rich domain. Using solid phase binding assays and antibody inhibition experiments, we demonstrated that the recombinant disintegrin-cysteine-rich domain of ADAM-9 specifically interacts with the beta1 integrin subunit on keratinocytes. This was corroborated by co-immunoprecipitation. In addition, engagement of integrin receptors by the disintegrin-cysteine-rich domain resulted in ERK phosphorylation and increased MMP-9 synthesis. Treatment with the ERK inhibitor PD98059 inhibited MMP-9 induction. Furthermore, the presence of the soluble disintegrin-cysteine-rich domain did not interfere with cell migration on different substrates. However, keratinocytes adhering to the immobilized disintegrin-cysteine-rich domain showed increased motility, which was partially due to the induction of MMP-9 secretion. In summary, our results indicate that the ADAM-9 adhesive domain plays a role in regulating the motility of cells by interaction with beta1 integrins and modulates MMP synthesis.

Melanoma cell-derived vascular endothelial growth factor induces endothelial tubulogenesis within fibrin gels by a metalloproteinase-mediated mechanism.

Angiogenesis is a process required not only for embryonal development but is encountered in wound healing and in pathological situations such as tumour growth. In vitro, formation of capillary-like structures can be induced by seeding human microvascular endothelial cells (HDMECs) on top of a fibrin matrix in the presence of phorbol 12-myristate 13-acetate (PMA) as a stimulating agent. In this study, we show that supernatants collected from high-invasive melanoma cells (BLM) induce the formation of tubular structures similar to PMA treatment whereas supernatants from low-invasive cells (WM164) did not. Analysis of proteins secreted into the supernatant of both melanoma cell lines identified differential expression of several pro-angiogenic proteins in high- and low-invasive melanoma cells. Vascular endothelial growth factor (VEGF) was strongly expressed by high- but not by low-invasive melanoma cells. Neutralisation of VEGF as well as inhibition of matrix metalloproteases (MMPs) using the broad spectrum MMP inhibitor 1,10-phenanthroline, both strongly reduced the melanoma-induced tube formation. PMA treatment of HDMECs on a fibrin matrix stimulated MT1-MMP synthesis, indicating that this protease is involved in PMA-induced angiogenesis. In addition, stimulation of HDMECs by supernatants of BLM melanoma cells resulted in a strong induction of ADAM-15, which is known to act as a metalloproteinase. In conclusion, these results show that VEGF released by melanoma cells is an important mediator of neo-vascularisation and that this process depends on the presence of metalloproteinases.