RESUMO
CUB domain-containing protein 1 (CDCP1) is an oncogenic orphan transmembrane receptor and a promising target for the detection and treatment of cancer. Extracellular proteolysis of CDCP1 by poorly defined mechanisms induces pro-metastatic signaling. We describe a new approach for the rapid identification of proteases responsible for key proteolytic events using a substrate-biased activity-based probe (sbABP) that incorporates a substrate cleavage motif grafted onto a peptidyl diphenyl phosphonate warhead for specific target protease capture, isolation and identification. Using a CDCP1-biased probe, we identify urokinase (uPA) as the master regulator of CDCP1 proteolysis, which acts both by directly cleaving CDCP1 and by activating CDCP1-cleaving plasmin. We show that coexpression of uPA and CDCP1 is strongly predictive of poor disease outcome across multiple cancers and demonstrate that uPA-mediated CDCP1 proteolysis promotes metastasis in disease-relevant preclinical in vivo models. These results highlight CDCP1 cleavage as a potential target to disrupt cancer and establish sbABP technology as a new approach to identify disease-relevant proteases.
Assuntos
Antígenos de Neoplasias/metabolismo , Moléculas de Adesão Celular/metabolismo , Peptídeo Hidrolases/análise , Animais , Antígenos de Neoplasias/química , Antígenos de Neoplasias/genética , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/genética , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Estrutura Molecular , Peptídeo Hidrolases/metabolismo , Especificidade por SubstratoRESUMO
BACKGROUND: The small GTP-binding protein Rab31 plays an important role in the modulation of tumor biological-relevant processes, including cell proliferation, adhesion, and invasion. As an underlying mechanism, Rab31 is presumed to act as a molecular switch between a more proliferative and an invasive phenotype. This prompted us to analyze whether Rab31 overexpression in breast cancer cells affects expression of genes involved in epithelial-to-mesenchymal transition (EMT)-like processes when compared to Rab31 low-expressing cells. METHODS: Commercially available profiler PCR arrays were applied to search for differentially expressed genes in Rab31 high- and low-expressing CAMA-1 breast cancer cells. Differential expression of selected candidate genes in response to Rab31 overexpression in CAMA-1 cells was validated by independent qPCR and protein assays. RESULTS: Gene expression profiling of key genes involved in EMT, or its reciprocal process MET, identified 9 genes being significantly up- or down-regulated in Rab31 overexpressing CAMA-1 cells, with the strongest effects seen for TGFB1, encoding TGF-ß1 (> 25-fold down-regulation in Rab31 overexpressing cells). Subsequent validation analyses by qPCR revealed a strong down-regulation of TGFB1 mRNA levels in response to increased Rab31 expression not only in CAMA-1 cells, but also in another breast cancer cell line, MDA-MB-231. Using ELISA and Western blot analysis, a considerable reduction of both intracellular and secreted TGF-ß1 antigen levels was determined in Rab31 overexpressing cells compared to vector control cells. Furthermore, reduced TGF-ß activity was observed upon Rab31 overexpression in CAMA-1 cells using a sensitive TGF-ß bioassay. Finally, the relationship between Rab31 expression and the TGF-ß axis was analyzed by another profiler PCR array focusing on genes involved in TGF-ß signaling. We found 12 out of 84 mRNAs significantly reduced and 7 mRNAs significantly increased upon Rab31 overexpression. CONCLUSIONS: Our results demonstrate that Rab31 is a potent modulator of the expression of TGF-ß and other components of the TGF-ß signaling pathway in breast cancer cells.
Assuntos
Neoplasias da Mama/genética , Transição Epitelial-Mesenquimal/genética , Fator de Crescimento Transformador beta1/genética , Proteínas rab de Ligação ao GTP/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Fator de Crescimento Transformador beta1/metabolismo , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Triple-negative breast cancer (TNBC), lacking the steroid hormone receptors ER and PR and the oncoprotein HER2, is characterized by its aggressive pattern and insensitivity to endocrine and HER2-directed therapy. Human kallikrein-related peptidases KLK1-15 provide a rich source of serine protease-type biomarkers associated with tumor growth and cancer progression for a variety of malignant diseases. In this study, recombinant KLK4 protein was generated and affinity-purified KLK4-directed polyclonal antibody pAb587 established to allow localization of KLK4 protein expression in tumor cell lines and archived formalin-fixed, paraffin-embedded TNBC tumor tissue specimens. For this, KLK4 protein expression was assessed by immunohistochemistry in primary tumor tissue sections (tissue microarrays) of 188 TNBC patients, mainly treated with anthracycline- or CMF-based polychemotherapy. KLK4 protein is localized in the cytoplasm of tumor and stroma cells. In this patient cohort, elevated stroma cell KLK4 expression, but not tumor cell KLK4 expression, is predictive for poor disease-free survival by univariate analysis (hazard ratio: 2.26, p=0.001) and multivariable analysis (hazard ratio: 2.12, p<0.01). Likewise, univariate analysis revealed a trend for statistical significance of elevated KLK4 stroma cell expression for overall survival of TNBC patients as well.
Assuntos
Biomarcadores Tumorais/metabolismo , Calicreínas/metabolismo , Neoplasias de Mama Triplo Negativas/enzimologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais/biossíntese , Humanos , Calicreínas/biossíntese , Análise Multivariada , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/patologia , Células Tumorais CultivadasRESUMO
BACKGROUND: Tumour-infiltrating lymphocytes (TILs) are associated with improved survival in several epithelial cancers. The two chemokines CXCL9 and CXCL10 facilitate chemotactic recruitment of TILs, and their intratumoral accumulation is a conceivable way to improve TIL-dependent immune intervention in cancer. However, the prognostic impact of CXCL9 and CXCL10 in high-grade serous ovarian cancer (HGSC) is largely unknown. METHODS: One hundred and eighty four cases of HGSC were immunohistochemically analyzed for CXCL9, CXCL10. TILs were assessed using CD3, CD56 and FOXP3 staining. Chemokine regulation was investigated using the ovarian cancer cell lines OV-MZ-6 and SKOV-3. RESULTS: High expression of CXCL9 and CXCL10 was associated with an approximately doubled overall survival (n=70, CXCL9: HR 0.41; P=0.006; CXCL10: HR 0.46; P=0.010) which was confirmed in an independent validation set (n=114; CXCL9: HR 0.60; P=0.019; CXCL10: HR 0.52; P=0.005). Expression of CXCR3 ligands significantly correlated with TILs. In human ovarian cancer cell lines the cyclooxygenase (COX) metabolite Prostaglandin E2 was identified as negative regulator of chemokine secretion, whereas COX inhibition by indomethacin significantly upregulated CXCL9 and CXCL10. In contrast, celecoxib, the only COX inhibitor prospectively evaluated for therapy of ovarian cancer, suppressed NF-κB activation and inhibited chemokine release. CONCLUSION: Our results support the notion that CXCL9 and CXCL10 exert tumour-suppressive function by TIL recruitment in human ovarian cancer. COX inhibition by indomethacin, not by celecoxib, may be a promising approach to concomitantly improve immunotherapies.
Assuntos
Quimiocina CXCL10/fisiologia , Quimiocina CXCL9/fisiologia , Inibidores de Ciclo-Oxigenase/farmacologia , Neoplasias Ovarianas/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Indometacina/farmacologia , Ligantes , Neoplasias Ovarianas/patologia , Prognóstico , Análise de SobrevidaRESUMO
The αvß6 integrin binds the RGD-containing peptide of the foot and mouth disease virus with high selectivity. In this study, the long binding helix of this ligand was downsized to an enzymatically stable cyclic peptide endowed with sub-nanomolar binding affinity toward the αvß6 receptor and remarkable selectivity against other integrins. Computational studies were performed to disclose the molecular bases underlying the high binding affinity and receptor subtype selectivity of this peptide. Finally, the utility of the ligand for use in biomedical studies was also demonstrated here.
Assuntos
Antígenos de Neoplasias/química , Integrinas/química , Peptídeos/química , Sequência de Aminoácidos , Linhagem Celular Tumoral , Humanos , Ligantes , Espectroscopia de Ressonância Magnética , Simulação de Dinâmica MolecularRESUMO
The tetraspanin CD63 is implicated in pro-metastatic signaling pathways but, so far, it is unclear, how CD63 levels affect the tumor cell phenotype. Here, we investigated the effect of CD63 modulation in different metastatic tumor cell lines. In vitro, knock down of CD63 induced a more epithelial-like phenotype concomitant with increased E-cadherin expression, downregulation of its repressors Slug and Zeb1, and decreased N-cadherin. In addition, ß-catenin protein was markedly reduced, negatively affecting expression of the target genes MMP-2 and PAI-1. ß-catenin inhibitors mimicked the epithelial phenotype induced by CD63 knock down. Inhibition of ß-catenin upstream regulators PI3K/AKT or GSK3ß could rescue the mesenchymal phenotype underlining the importance of the ß-catenin pathway in CD63-regulated cell plasticity. CD63 knock down-induced phenotypical changes correlated with a decrease of experimental metastasis whereas CD63 overexpression enhanced the tumor cell-intrinsic metastatic potential. Taken together, our data show that CD63 is a crucial player in the regulation of the tumor cell-intrinsic metastatic potential by affecting cell plasticity.
Assuntos
Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Tetraspanina 30/metabolismo , beta Catenina/metabolismo , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Dados de Sequência Molecular , Transdução de Sinais , Tetraspanina 30/genéticaRESUMO
Integrins, as transmembrane heterodimeric receptors, have important functions in cell adhesion, migration, proliferation, survival apoptosis and signal transduction, in many physio- as well as pathophysiological settings. Characterisation of integrins and their ligand/antagonist binding is notoriously difficult, due to high integrin redundancy and ubiquity. Bypassing the intrinsic difficulties of cell-based integrin expression, purification and reconstitution, we present for the first time the synthesis of a heterodimeric integrin receptor and its assembly into a block-copolymeric membrane mimic. We present comprehensive data to demonstrate the synthesis of functionally active integrin αv ß3, generated by in vitro membrane-assisted protein synthesis (iMAPS). This work represents the first step towards a robust and adaptable polymer-based platform for characterisation of integrin-ligand interactions.
Assuntos
Integrinas/metabolismo , Modelos Moleculares , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Adesão Celular , Sistema Livre de Células , Integrinas/química , Microscopia Confocal , Estrutura Molecular , Fosfatidilcolinas/síntese química , Fosfatidilcolinas/química , Complexo Glicoproteico GPIb-IX de Plaquetas/química , Dobramento de ProteínaRESUMO
Despite great efforts in improving existing therapies, the outcome of patients with advanced radioresistant HPV-negative head and neck squamous cell carcinoma (HNSCC) remains poor. The chromatin remodeler Chromodomain helicase DNA binding protein 4 (CHD4) is involved in different DNA-repair mechanisms, but the role and potential in HNSCC has not been explored yet. In the present study, we evaluated the prognostic significance of CHD4 expression using in silico analysis of the pan-cancer dataset. Furthermore, we established a monoclonal HNSCC CHD4 knockdown cell clone utilizing the CRISPR/Cas9 system. Effects of lower CHD4 expression on radiosensitivity after increasing doses of ionizing radiation were characterized using clonogenic assays and cell numbers. The in silico analysis revealed that high CHD4 expression is associated with significant poorer overall survival of HPV-negative HNSCC patients. Additionally, the knockdown of CHD4 significantly increased the radiosensitivity of HNSCC cells. Therefore, CHD4 might be involved in promoting radioresistance in hard-to-treat HPV-negative HNSCC entities. We conclude that CHD4 could serve as a prognostic factor in HPV-negative HNSCC tumors and is a potential target protein overcoming radioresistance in HNSCC. Our results and the newly established cell clone laid the foundation to further characterize the underlying mechanisms and ultimately use CHD4 in HNSCC therapies.
Assuntos
Neoplasias de Cabeça e Pescoço , Infecções por Papillomavirus , Humanos , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/radioterapia , Carcinoma de Células Escamosas de Cabeça e Pescoço/complicações , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/radioterapia , Neoplasias de Cabeça e Pescoço/complicações , Prognóstico , Linhagem Celular Tumoral , Complexo Mi-2 de Remodelação de Nucleossomo e DesacetilaseRESUMO
BACKGROUND: The treatment of oral squamous cell carcinoma (OSCC) remains challenging and survival rates have not been improved significantly over the past decades. Integrins have been recognized driving the cancer progression and high expression levels cause poor outcomes in patients afflicted with OSCC. Integrin αvß6 and its subunit integrin beta 6 (ITGB6) were discovered to enhance the invasiveness by providing beneficial effects on downstream pathways promoting the cancer progression. The objective of this study was to establish a CRISPR/Cas9-mediated knock out of ITGB6 in the human OSCC cell line HN and investigate the effects on the migration and proliferation ability. METHODS: ITGB6 knock out was performed using the CRISPR/Cas9-system, RNPs, and lipofection. Monoclonal cell clones were achieved by limiting dilution and knock out verification was carried out by sanger sequencing and FACS on protein level. The effects of the knock out on the proliferation and migration ability were evaluated by using MTT and scratch assays. In addition, in silico TCGA analysis was utilized regarding the effects of ITGB6 on overall survival and perineural invasion. RESULTS: In silico analysis revealed a significant impact of ITGB6 mRNA expression levels on the overall survival of patients afflicted with OSCC. Additionally, a significantly higher rate of perineural invasion was discovered. CRISPR/Cas9-mediated knock out of ITGB6 was performed in the OSCC cell line HN, resulting in the generation of a monoclonal knock out clone. The knock out clone exhibited a significantly reduced migration and proliferation ability when compared to the wildtype. CONCLUSIONS: ITGB6 is a relevant factor in the progression of OSCC and can be used for the development of novel treatment strategies. The present study is the first to establish a monoclonal CRISPR/Cas9-mediated ITGB6 knockout cell clone derived from an OSCC cell line. It suggests that ITGB6 has a significant impact on the proliferative and migratory capacity in vitro.
Assuntos
Sistemas CRISPR-Cas , Carcinoma de Células Escamosas , Movimento Celular , Proliferação de Células , Cadeias beta de Integrinas , Neoplasias Bucais , Humanos , Movimento Celular/genética , Proliferação de Células/genética , Neoplasias Bucais/genética , Neoplasias Bucais/patologia , Linhagem Celular Tumoral , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Cadeias beta de Integrinas/genética , Técnicas de Inativação de Genes , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Invasividade Neoplásica/genética , Regulação Neoplásica da Expressão GênicaRESUMO
Dysregulated apoptotic pathways are regarded as major reasons for chemoresistance development as a particular challenge in ovarian cancer therapy. In search of molecular factors affecting human ovarian cancer cell apoptosis and, consequently, patient survival, we examined tumors of 103 platinum-/taxane-treated ovarian cancer patients by mRNA-array hybridization, qPCR, and immunohistochemistry. We identified high expression of crystallin αB (CRYAB), a proposed negative regulator of tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-mediated apoptosis. By Kaplan Meier analysis, this factor turned out to be significantly associated with poor patient outcome [overall survival (OS) p = 0.001, recurrence-free survival (RFS) p = 0.003]. Elevated hazard ratios (HR) were estimated with regard to OS (HR = 2.11, 95% CI 1.10-4.06) and RFS (HR = 1.92, 95% CI 1.07-3.47) in multivariable analyses. These associations were confirmed in independent, publicly available mRNA data comprising 431 patients for OS (p < 0.001) and 413 for RFS (p < 0.001). Our findings were validated by studying apoptotic events in cultured human ovarian cancer cells which were stably transfected to express elevated CRYAB levels. These data emphasized the crucial role of CRYAB in human ovarian cancer biology since TRAIL- as well as cisplatin-induced apoptosis was significantly impaired as a function of enhanced CRYAB expression. Taken together, we identified CRYAB as an independent biomarker for unfavourable outcome of human ovarian cancer patients. Since TRAIL is currently tested as anti-cancer drug and large proportions of the present patient cohort displayed low CRYAB levels in their tumors, CRYAB may enable the selection of patient subgroups benefiting most from TRAIL-containing therapy.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Biomarcadores Tumorais/genética , Cisplatino/farmacologia , Neoplasias Ovarianas/genética , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Cadeia B de alfa-Cristalina/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/metabolismo , Caspase 3/metabolismo , Linhagem Celular Tumoral , Ativação Enzimática/efeitos dos fármacos , Feminino , Humanos , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Neoplasias Ovarianas/mortalidade , Neoplasias Ovarianas/patologia , Prognóstico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Cadeia B de alfa-Cristalina/metabolismoRESUMO
Pattern seekers: For the two angiogenic relevant integrins α5ß1 and αvß3, functionalized derivatives of the selective antagonists 1 and 2 could target and discriminate between tumor cells inâ vivo based on their different integrin patterns and also delay tumor growth inâ vivo. In addition, the first α5ß1-selective integrin antagonist that enables specific molecular imaging by positron emission tomography was developed.
Assuntos
Integrina alfa5beta1/antagonistas & inibidores , Integrina alfaVbeta3/antagonistas & inibidores , Tomografia por Emissão de Pósitrons/métodos , Moduladores da Angiogênese , Animais , Humanos , Camundongos , Peptidomiméticos , RatosRESUMO
Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with poor patient prognosis and limited therapeutic options. A lack of prognostic biomarkers and therapeutic targets fuels the need for new approaches to tackle this severe disease. Extracellular matrix degradation, release, and modulation of the activity of growth factors/cytokines/chemokines, and the initiation of signaling pathways by extracellular proteolytic networks, have been identified as major processes in the carcinogenesis of breast cancer. Members of the kallikrein-related peptidase (KLK) family contribute to these tumor-relevant processes, and are associated with breast cancer progression and metastasis. In this study, the clinical relevance of mRNA expression of two members of this family, KLK10 and KLK11, has been evaluated in TNBC. For this, their expression levels were quantified in tumor tissue of a large, well-characterized patient cohort (n = 123) via qPCR. Although, in general, the overall expression of both factors are lower in tumor tissue of breast cancer patients (encompassing all subtypes) compared to normal tissue of healthy donors, in the TNBC subtype, expression is even increased. In our cohort, a significant, positive correlation between the expression levels of both KLKs was detected, indicating a coordinate expression mode of these proteases. Elevated KLK10 and KLK11 mRNA levels were associated with poor patient prognosis. Moreover, both factors were found to be independent of other established clinical factors such as age, lymph node status, or residual tumor mass, as determined by multivariable Cox regression analysis. Thus, both proteases, KLK10 and KLK11, may represent unfavorable prognostic factors for TNBC patients and, furthermore, appear as promising potential targets for therapy in TNBC.
RESUMO
We previously showed that integrin αvß3 expression upon engagement by its major ligand vitronectin (VN) correlated with enhanced human ovarian cancer cell adhesion, motility, and proliferation, by triggering intracellular signaling events, ultimately leading to altered gene expression. In the present study, we characterized cellular VN expression as a function of αvß3 and noticed significant upregulation of VN protein which was reflected by elevated VN gene transcription. In order to identify specific transcription factors involved in the αvß3-regulatory effect on VN, we generated different VN promoter mutants. We noticed that disruption of the DNA consensus motif for Rel proteins did not only prominently reduce VN promoter activity but, moreover, led to a loss of responsiveness to αvß3, suggesting a crucial role of Rel proteins in αvß3-provoked VN induction. In cell migration studies, we confirmed increased cell motility as a function of αvß3/VN which was further enhanced by raising cellular Rel transcription factor levels. Thus, the data of the present study elucidated a positive feedback regulatory loop on VN expression by αvß3 implicating transcription factors of the Rel family. Hence by altering the composition of the extracellular matrix upon additional VN synthesis and deposition, tumor cells might be enabled to modulate their surrounding reactive microenvironment towards enhanced αvß3/VN-interactions and, consequently, intrinsic intracellular signaling events affecting cancer progression.
Assuntos
Integrina alfaVbeta3/metabolismo , Proteínas Proto-Oncogênicas c-rel/metabolismo , Proteínas Recombinantes/metabolismo , Vitronectina/genética , Sequência de Bases , Linhagem Celular Tumoral , Movimento Celular/genética , Retroalimentação Fisiológica , Feminino , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genes Reporter , Humanos , Integrina alfaVbeta3/genética , Luciferases de Renilla/genética , Luciferases de Renilla/metabolismo , Dados de Sequência Molecular , Neoplasias Ovarianas , Regiões Promotoras Genéticas , Proteínas Recombinantes/genética , Regulação para Cima , Vitronectina/metabolismoRESUMO
uPAR, the three-domain membrane receptor of the serine protease urokinase, plays a crucial role in tumor growth and metastasis. Several mRNA splice variants of this receptor have been reported. One of these, uPAR-del4/5, lacking exons 4 and 5, and thus encoding a uPAR form lacking domain DII, is specifically overexpressed in breast cancer and represents a statistically independent prognostic factor for distant metastasis-free survival in breast cancer patients. The aim of the present study was to examine the molecular and cellular properties of the encoded uPAR-del4/5 protein. To investigate the impact of the uPAR-del4/5 overexpression on in vitro and in vivo aspects of tumor progression (e.g., proliferation, adhesion, invasion, metastatic seeding, and/or metastatic growth), we combined the analysis of transfected cancer cell lines with a murine xenograft tumor model. Increased expression of uPAR-del4/5 in human cancer cells led to reduced adhesion to several extracellular matrix proteins and decreased invasion through Matrigel, while cell proliferation was not affected in vitro. Moreover, invasion of uPAR-del4/5 overexpressing cells was not altered by addition of urokinase, while that of uPAR-wild-type overexpressing cells was drastically increased. Accordingly, we observed that, in contrast to uPAR-wild-type, uPAR-del4/5 does not interact with urokinase. On the other hand, when overexpressed in human breast cancer cells, uPAR-del4/5 distinctly impaired metastatic dissemination and growth in vivo. We demonstrate that the uPAR-del4/5 mRNA splice variant mediates tumor-relevant biological processes in vitro and in vivo. Our results thus illustrate how tumor-specific alternative splicing can distinctly impact the biology of the tumor.
Assuntos
Processamento Alternativo , Neoplasias da Mama/genética , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Animais , Western Blotting , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , Ensaio de Imunoadsorção Enzimática , Feminino , Imunofluorescência , Humanos , Camundongos , Invasividade Neoplásica , Metástase Neoplásica , Transplante de Neoplasias , RNA Mensageiro/genética , Deleção de SequênciaRESUMO
Vitronectin is an abundant adhesive glycoprotein in blood plasma and is found associated with different extracellular matrix sites, the vessel wall, and tumor cells, particularly upon tissue remodeling, injury/repair, or under disease conditions. Plasma vitronectin is a structurally labile molecule that may be converted into a multimeric/multivalent form by interaction with various (hemostatic) factors or through surface binding. Several distinct binding domains along the vitronectin sequence for integrin-type cell adhesion receptors, for urokinase receptor or proteoglycans as well as for growth factors, endow vascular matrix- or fibrin-associated vitronectin with differentiated cell attachment and aggregatory properties. These were found to be relevant for modulation of the cell-matrix interface in angiogenesis, hemostasis and thrombus formation, or wound repair, respectively. Other vitronectin ligands include plasminogen activator inhibitor (PAI)-1 or high molecular weight kininogen that confer strong antiadhesive functions upon integrin- or urokinase receptor-mediated cell interactions with vitronectin. Together, vitronectin acts as a potent matricellular factor, coordinating cell migration with pericellular proteolysis and growth factor signaling at sites of tissue remodeling or in tumors. Structure-function studies of such vitronectin-related ligands and receptors lead to the characterization of their mode of action, also stimulating the search for new antagonists in tumor angiogenesis, platelet aggregation, or atherosclerosis. This review focuses on new developments in vitronectin biology, with particular emphasis on regulatory mechanisms of the protein in the context of cell adhesion/migration/proliferation and cell-dependent proteolysis, relevant for our understanding of hemostasis, thrombosis, tissue repair, and vascular diseases.
Assuntos
Vitronectina/fisiologia , Animais , Coagulação Sanguínea/fisiologia , Vasos Sanguíneos/fisiologia , Hemostasia , Humanos , Vitronectina/sangue , Vitronectina/químicaRESUMO
Integrins have been extensively investigated as therapeutic targets over the last decades, which has been inspired by their multiple functions in cancer progression, metastasis, and angiogenesis as well as a continuously expanding number of other diseases, e.g., sepsis, fibrosis, and viral infections, possibly also Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2). Although integrin-targeted (cancer) therapy trials did not meet the high expectations yet, integrins are still valid and promising targets due to their elevated expression and surface accessibility on diseased cells. Thus, for the future successful clinical translation of integrin-targeted compounds, revisited and innovative treatment strategies have to be explored based on accumulated knowledge of integrin biology. For this, refined approaches are demanded aiming at alternative and improved preclinical models, optimized selectivity and pharmacological properties of integrin ligands, as well as more sophisticated treatment protocols considering dose fine-tuning of compounds. Moreover, integrin ligands exert high accuracy in disease monitoring as diagnostic molecular imaging tools, enabling patient selection for individualized integrin-targeted therapy. The present review comprehensively analyzes the state-of-the-art knowledge on the roles of RGD-binding integrin subtypes in cancer and non-cancerous diseases and outlines the latest achievements in the design and development of synthetic ligands and their application in biomedical, translational, and molecular imaging approaches. Indeed, substantial progress has already been made, including advanced ligand designs, numerous elaborated pre-clinical and first-in-human studies, while the discovery of novel applications for integrin ligands remains to be explored.
RESUMO
Epithelial ovarian cancer involves the shedding of single tumor cells or spheroids from the primary tumor into ascites, followed by their survival, and transit to the sites of metastatic colonization within the peritoneal cavity. During their flotation, anchorage-dependent epithelial-type tumor cells gain anoikis resistance, implicating integrins, including αvß3. In this study, we explored anoikis escape, cisplatin resistance, and prosurvival signaling as a function of the αvß3 transmembrane conformational activation state in cells suspended in ascites. A high-affinity and constitutively signaling-competent αvß3 variant, which harbored unclasped transmembrane domains, was found to confer delayed anoikis onset, enhanced cisplatin resistance, and reduced cell proliferation in ascites or 3D-hydrogels, involving p27kip upregulation. Moreover, it promoted EGF-R expression and activation, prosurvival signaling, implicating FAK, src, and PKB/Akt. This led to the induction of the anti-apoptotic factors Bcl-2 and survivin suppressing caspase activation, compared to a signaling-incapable αvß3 variant displaying firmly associated transmembrane domains. Dissecting the mechanistic players for αvß3-dependent survival and peritoneal metastasis of ascitic ovarian cancer spheroids is of paramount importance to target their anchorage independence by reversing anoikis resistance and blocking αvß3-triggered prosurvival signaling.
Assuntos
Anoikis , Integrina alfaVbeta3/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Ovarianas/metabolismo , Transdução de Sinais , Linhagem Celular Tumoral , Feminino , Humanos , Integrina alfaVbeta3/genética , Proteínas de Neoplasias/genética , Neoplasias Ovarianas/genéticaRESUMO
High levels of reactive oxygen species (ROS) in tumors have been shown to exert anti-tumor activity, leading to the concept of ROS induction as therapeutic strategy. The organometallic compound ferrocene (Fc) generates ROS through a reversible one-electron oxidation. Incorporation of Fc into a tumor-targeting, bioactive molecule can enhance its therapeutic activity and enable tumor specific delivery. Therefore, we conjugated Fc to five synthetic, Arg-Gly-Asp (RGD)-based integrin binding ligands to enable targeting of the cell adhesion and signaling receptor integrin subtypes αvß3, α5ß1, or αvß6, which are overexpressed in various, distinct tumors. We designed and synthesized a library of integrin-ligand-ferrocene (ILF) derivatives and showed that ILF conjugates maintained the high integrin affinity and selectivity of their parent ligands. A thorough biological characterization allowed us to identify the two most promising ligands, an αvß3 (L2b) and an αvß6 (L3b) targeting ILF, which displayed selective integrin-dependent cell uptake and pronounced ferrocene-mediated anti-tumor effects in vitro, along with increased ROS production and DNA damage. Hence, ILFs are promising candidates for the selective, tumor-targeted delivery of ferrocene to maximize its anti-cancer efficacy and minimize systemic toxicity, thereby improving the therapeutic window of ferrocene compared to currently used non-selective anti-cancer drugs.
Assuntos
Integrinas , Neoplasias , Humanos , Integrina alfa5beta1 , Integrina alfaVbeta3 , Ligantes , Metalocenos , Neoplasias/tratamento farmacológicoRESUMO
A crucial mode of action of trastuzumab is the labeling of HER2-positive (HER2+) tumor cells for the eradication by natural killer (NK) cells, a process called antibody-dependent cellular cytotoxicity (ADCC). However, despite widespread HER2 expression among cancer entities, only a fraction, with robust HER2 overexpression, benefits from trastuzumab therapy. ADCC requires both sufficient lymphocytic infiltration and close binding of the immune cells to the antibody-tagged tumor cells. We hypothesized that the chemokine CX3CL1 could improve both processes, as it is synthesized as a membrane-bound, adhesive form that is eventually cleaved into a soluble, chemotactic protein. Here, we show that CX3CL1 overexpression is a positive prognostic marker in breast cancer. CX3CL1 overexpression attracted tumor-suppressive lymphocytes, including NK cells, and inhibited tumor growth and lung metastasis in the syngeneic 4T1 breast cancer mouse model. In HER2+ SKBR3, MDA-MB-453, and HT-29 tumor cells, CX3CL1 overexpression increased NK cell-mediated cytotoxicity in vitro and acted synergistically with trastuzumab. Even though CX3CL1 did not further improve trastuzumab efficacy in vivo in the trastuzumab-sensitive MDA-MB-453 model, it compensated for NK-cell depletion and prolonged survival. In the HER2 low-expressing HT-29 model, however, CX3CL1 overexpression not only prolonged survival time but also overcame trastuzumab resistance in a partly NK cell-dependent manner. Taken together, these findings identify CX3CL1 as a feasible pharmacologic target to enable trastuzumab therapy in HER2 low-expressing cancers and render it a potential predictive biomarker to determine therapy responders.
Assuntos
Antineoplásicos Imunológicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Quimiocina CX3CL1/genética , Neoplasias Pulmonares/tratamento farmacológico , Trastuzumab/farmacologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antineoplásicos Imunológicos/uso terapêutico , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Quimiocina CX3CL1/metabolismo , Estudos de Coortes , Resistencia a Medicamentos Antineoplásicos/genética , Resistencia a Medicamentos Antineoplásicos/imunologia , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/imunologia , Humanos , Estimativa de Kaplan-Meier , Células Matadoras Naturais/imunologia , Neoplasias Pulmonares/secundário , Camundongos , Pessoa de Meia-Idade , Prognóstico , Receptor ErbB-2/análise , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/metabolismo , Transdução de Sinais/imunologia , Trastuzumab/uso terapêutico , Microambiente Tumoral/genética , Microambiente Tumoral/imunologia , Adulto JovemRESUMO
The tetraspanin KAI1 had been described as a metastasis suppressor in many different cancer types, a function for which associations of KAI1 with adhesion and signaling receptors of the integrin superfamily likely play a role. In ovarian cancer, integrin alphavbeta3 correlates with tumor progression and its elevation in vitro provoked enhanced cell adhesion accompanied by significant increases in cell motility and proliferation in the presence of its major ligand vitronectin. In the present study, we characterized integrin alphavbeta3-mediated tumor biological effects as a function of cellular KAI1 restoration and proved for the first time that KAI1, besides its already known physical crosstalk with beta1-integrins, also colocalizes with integrin alphavbeta3. Functionally, elevated KAI1 levels drastically increased integrin alphavbeta3/vitronectin-dependent ovarian cancer cell adhesion. Since an intermediate level of cell adhesive strength is required for optimal cell migration, we next studied ovarian cancer cell motility as a function of KAI1 restoration. By time lapse video microscopy, we found impaired integrin alphavbeta3/vitronectin-mediated cell migration most probably due to strongly enhanced cellular immobilization onto the adhesion-supporting matrix. Moreover, KAI1 reexpression significantly diminished cell proliferation. These data strongly indicate that KAI1 may suppress ovarian cancer progression by inhibiting integrin alphavbeta3/vitronectin-provoked tumor cell motility and proliferation as important hallmarks of the oncogenic process.