RESUMO
Recent experimental evidence suggests that mebendazole, a popular antiparasitic drug, binds to heat shock protein 90 (Hsp90) and inhibits acute myeloid leukemia cell growth. In this study we use quantum mechanics (QM), molecular similarity, and molecular dynamics (MD) calculations to predict possible binding poses of mebendazole to the adenosine triphosphate (ATP) binding site of Hsp90. Extensive conformational searches and minimization of the five mebendazole tautomers using the MP2/aug-cc-pVTZ theory level resulted in 152 minima. Mebendazole-Hsp90 complex models were subsequently created using the QM optimized conformations and protein coordinates obtained from experimental crystal structures that were chosen through similarity calculations. Nine different poses were identified from a total of 600 ns of explicit solvent, all-atom MD simulations using two different force fields. All simulations support the hypothesis that mebendazole is able to bind to the ATP binding site of Hsp90.
Assuntos
Mebendazol , Simulação de Dinâmica Molecular , Trifosfato de Adenosina , Proteínas de Choque Térmico HSP90/química , Humanos , Mebendazol/farmacologia , Conformação Molecular , Ligação Proteica , Conformação ProteicaRESUMO
Modern cancer therapies increased the survival rates of acute myeloid leukemia (AML) patients tremendously. However, the complexity of the disease and the identification of new targets require the adaptation of treatment protocols to reduce side effects and increase benefit for the patients. One key regulator of leukemogenesis and chemotherapy resistance in AML is the Hedgehog (HH) signaling pathway. It is deregulated in numerous cancer entities and inhibition of its downstream transcription factors GLI translates into anti-leukemic effects. One major regulator of GLI is BRD4, a BET family member with epigenetic functions. We investigated the effect of ZEN-3365, a novel BRD4 inhibitor, on AML cells in regard to the HH pathway. We show that ZEN-3365 alone or in combination with GANT-61 reduced GLI promoter activity, cell proliferation and colony formation in AML cell lines and primary cells. Our findings strongly support the evaluation of the BRD4 inhibitor ZEN-3365 as a new therapeutic option in AML.
Assuntos
Antineoplásicos/farmacologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas Hedgehog/metabolismo , Leucemia Mieloide Aguda/tratamento farmacológico , Fatores de Transcrição/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Leucemia Mieloide Aguda/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/metabolismoRESUMO
The prognosis of elderly AML patients is still poor due to chemotherapy resistance. The Hedgehog (HH) pathway is important for leukemic transformation because of aberrant activation of GLI transcription factors. MBZ is a well-tolerated anthelmintic that exhibits strong antitumor effects. Herein, we show that MBZ induced strong, dose-dependent anti-leukemic effects on AML cells, including the sensitization of AML cells to chemotherapy with cytarabine. MBZ strongly reduced intracellular protein levels of GLI1/GLI2 transcription factors. Consequently, MBZ reduced the GLI promoter activity as observed in luciferase-based reporter assays in AML cell lines. Further analysis revealed that MBZ mediates its anti-leukemic effects by promoting the proteasomal degradation of GLI transcription factors via inhibition of HSP70/90 chaperone activity. Extensive molecular dynamics simulations were performed on the MBZ-HSP90 complex, showing a stable binding interaction at the ATP binding site. Importantly, two patients with refractory AML were treated with MBZ in an off-label setting and MBZ effectively reduced the GLI signaling activity in a modified plasma inhibitory assay, resulting in a decrease in peripheral blood blast counts in one patient. Our data prove that MBZ is an effective GLI inhibitor that should be evaluated in combination to conventional chemotherapy in the clinical setting.
Assuntos
Leucemia Mieloide Aguda/metabolismo , Mebendazol/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Moduladores de Tubulina/farmacologia , Proteína GLI1 em Dedos de Zinco/metabolismo , Estudos de Casos e Controles , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Humanos , Leucemia Mieloide Aguda/etiologia , Leucemia Mieloide Aguda/patologia , Leucemia Mieloide Aguda/terapia , Proteólise , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Proteína GLI1 em Dedos de Zinco/antagonistas & inibidores , Proteína GLI1 em Dedos de Zinco/químicaRESUMO
This study aimed to characterize different natural killer (NK) cell phenotypes on bone marrow and peripheral blood cells from acute myeloid leukemia (AML) patients and healthy donors (HDs). Our data show that CD56dimCD16- and CD56brightCD16- NK cells represent the predominant NK cell subpopulations in AML, while the CD56dimCD16+ NK cells are significantly reduced compared to HDs. Moreover, TIGIT+ and PVRIG+ cells cluster on the CD56dimCD16+ subset whereas CD39+ and CD38+ cells do so on CD56brightCD16- NK cells in AML. Furthermore, functional effects of (co-)blockade of TIGIT and CD39 or A2AR on NK cell functionality were analyzed. These experiments revealed that the single blockade of the TIGIT receptor results in an increased NK-92 cell-mediated killing of AML cells in vitro. Combined targeting of CD39 or A2AR significantly augments the anti-TIGIT-mediated lysis of AML cells. Our data indicate that distinct NK cell subsets in AML exhibit different immunosuppressive patterns (via the TIGIT/PVRIG receptors and the purinergic pathway). In summary, we conclude that TIGIT, CD39, and A2AR constitute relevant inhibitory checkpoints of NK cells in AML patients. A combinatorial blockade synergistically strengthens NK-92 cell-mediated cytotoxicity. As inhibitors of TIGIT, CD39, and A2AR are clinically available, studies on their combined use could be conducted in the near future.
Assuntos
Apirase/metabolismo , Células Matadoras Naturais/metabolismo , Leucemia Mieloide Aguda/imunologia , Receptor A2A de Adenosina/metabolismo , Receptores Imunológicos/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Apirase/antagonistas & inibidores , Estudos de Casos e Controles , Humanos , Imunoterapia , Leucemia Mieloide Aguda/terapia , Pessoa de Meia-Idade , Receptores Imunológicos/antagonistas & inibidores , Adulto JovemRESUMO
INTRODUCTION: Prostate cancer has a special predilection to form bone metastases. Despite the known impact of the microvascular network on tumour growth and its dependence on the organ-specific microenvironment, the characteristics of the tumour vasculature in bone remain unknown. METHODS: The cell lines LNCaP, DU145, and PC3 were implanted into the femurs of NSG mice to examine the microvascular properties of prostate cancer in bone. Tumour growth and the functional and morphological alterations of the microvasculature were analysed for 21 days in vivo using a transparent bone chamber and fluorescence microscopy. RESULTS: Vascular density was significantly lower in tumour-bearing bone than in non-tumour-bearing bone, with a marked loss of small vessels. Accelerated blood flow velocity led to increased volumetric blood flow per vessel, but overall perfusion was not affected. All of the prostate cancer cell lines had similar vascular patterns, with more pronounced alterations in rapidly growing tumours. Despite minor differences between the prostate cancer cell lines associated with individual growth behaviours, the same overall pattern was observed and showed strong similarity to that of tumours growing in soft tissue. DISCUSSION: The increase in blood flow velocity could be a specific characteristic of prostate cancer or the bone microenvironment.
Assuntos
Neoplasias Ósseas/irrigação sanguínea , Neoplasias Ósseas/secundário , Osso e Ossos/patologia , Neoplasias da Próstata/patologia , Microambiente Tumoral , Animais , Humanos , Microscopia Intravital , Masculino , Camundongos , Microcirculação , Células PC-3 , Distribuição AleatóriaRESUMO
Aberrant activation of the hedgehog (HH) pathway is observed in many neoplasms, including acute myeloid leukemia (AML). The glioma-associated oncogene homolog (GLI) transcription factors are the main downstream effectors of the HH signaling cascade and are responsible for the proliferation and maintenance of leukemic stem cells, which support chemotherapy resistance and leukemia relapse. Cytarabine (Ara-C)-resistant variants of AML cell lines were established through long-term cultivation with successively increasing Ara-C concentrations. Subsequently, differences in GLI expression were analyzed by RT-qPCR. GLI3 mRNA levels were detectable in parental Kasumi-1, OCI-AML3, and OCI-AML5 cells, whereas GLI3 expression was completely silenced in all resistant counterparts. Therefore, we generated GLI3-knockdown cell lines using small hairpin RNAs (shRNA) and evaluated their sensitivity to Ara-C in vitro. The knockdown of GLI3 partly abolished the effect of Ara-C on colony formation and induction of apoptosis, indicating that GLI3 downregulation results in Ara-C resistance. Moreover, we analyzed the expression of several genes involved in Ara-C metabolism and transport. Knockdown of GLI3 resulted in the upregulation of SAM and HD domain-containing protein 1 (SAMHD1), cytidine deaminase (CDA), and ATP-binding cassette C11 (ABCC11)/multidrug resistance-associated protein 8 (MRP8), each of which has been identified as a predictive marker for Ara-C response in acute myeloid leukemia. Our results demonstrate that GLI3 downregulation is a potential mechanism to induce chemotherapy resistance in AML.
Assuntos
Apoptose/genética , Citarabina/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Leucemia Mieloide Aguda/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Transdução de Sinais/genética , Proteína Gli3 com Dedos de Zinco/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Citarabina/metabolismo , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Regulação para Baixo , Técnicas de Silenciamento de Genes , Inativação Gênica , Humanos , Leucemia Mieloide Aguda/genética , Proteínas do Tecido Nervoso/genética , Proteína 1 com Domínio SAM e Domínio HD/genética , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína Gli3 com Dedos de Zinco/genéticaRESUMO
During the last few years, diverse studies have shown that tumors can actively interact with the lymphatic system and promote metastases development. In order to examine the molecular mechanisms involved in this interaction, we co-cultured tumor and lymphatic endothelial cells (LEC) and subsequently analyzed the molecular alterations of LECs. Therefore, LECs were co-cultivated with either a highly or weakly metastatic breast cancer cell line using contact (mixture) and non-contact (transwell) co-cultures. mRNA profiles from LECs were subsequently analyzed for genes specifically induced by highly metastatic tumor cells ("metastatic specific"). Among the up-regulated "metastatic specific" genes, we found candidates involved in cell cycle, cell adhesion and motility (BST2, E-selectin, and HMMR), cytokines (CCL7, CXCL6, CXCL1, and CSF2) and factors of the complement system (C1R, C3, and CFB). Among the down-regulated genes, we detected the hyaluronan receptor STAB2, angiogenic factor apelin receptor (APLNR), and the glycosylation enzyme MAN1A1. In an additional prostate cancer co-culture model, we could confirm a "metastatic specific" upregulation of E-selectin and CCL7 in LECs after interaction with the prostate cancer cell lines LNCAP (highly metastatic) and DU145 (weakly metastatic). These data allowed us to identify a set of genes regulated in LECs during in vitro communication with cancer cells, which might subsequently facilitate lymphatic metastasis.
Assuntos
Biomarcadores Tumorais/análise , Neoplasias da Mama/patologia , Linfangiogênese , Vasos Linfáticos/patologia , Neoplasias da Próstata/patologia , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Adesão Celular , Técnicas de Cocultura , Feminino , Perfilação da Expressão Gênica , Humanos , Metástase Linfática , Vasos Linfáticos/metabolismo , Masculino , Invasividade Neoplásica , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Células Tumorais CultivadasRESUMO
Inositol-1,4,5-trisphosphate 3-kinase-A (ITPKA) exhibits oncogenic activity in lung cancer cells by regulating Ins(1,4,5)P3-mediated calcium release and cytoskeletal dynamics. Since, in normal cells, ITPKA is mainly expressed in the brain, it is an excellent target for selected therapy of lung cancer. However, ITPKB is strongly expressed in normal lung tissues, but is down-regulated in lung cancer cells by miR-375, assuming that ITPKB might have tumor suppressor activity. In addition, ITPKB binds to F-actin making it likely that, similar to ITPKA, it controls actin dynamics. Thus, the treatment of ITPKA-expressing lung cancer with ITPKA inhibitors simultaneously inhibiting ITPKB may counteract the therapy. Based on these considerations, we analyzed if ITPKB controls actin dynamics and if the protein reduces aggressive progression of lung cancer cells. We found that ITPKB bundled F-actin in cell-free systems. However, the stable expression of ITPKB in H1299 lung cancer cells, exhibiting very low endogenous ITPKB expression, had no significant effect on the actin structure. In addition, our data show that ITPKB negatively controls transmigration of H1299 cells in vitro by blocking Ins(1,4,5)P3-mediated calcium release. On the other hand, colony formation was stimulated by ITPKB, independent of Ins(1,4,5)P3-mediated calcium signals. However, dissemination of H1299 cells from the skin to the lung in NOD scid gamma mice was not significantly affected by ITPKB expression. In summary, ITPKB does not affect the cellular actin structure and does not suppress dissemination of human lung cancer cells in mice. Thus, our initial hypotheses that ITPKB exhibits tumor suppressor activity could not be supported.
Assuntos
Actinas/metabolismo , Neoplasias Pulmonares/enzimologia , Proteínas de Neoplasias/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Actinas/genética , Anticorpos Heterófilos , Linhagem Celular Tumoral , Sistema Livre de Células/metabolismo , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , MicroRNAs/genética , MicroRNAs/metabolismo , Metástase Neoplásica , Proteínas de Neoplasias/genética , Transplante de Neoplasias , Fosfotransferases (Aceptor do Grupo Álcool)/genética , RNA Neoplásico/genética , RNA Neoplásico/metabolismoRESUMO
Avoiding immune surveillance and inducing a tumor-promoting inflammatory milieu found entry into the new generation of the hallmarks of cancer. Cancer cells hijack immune mechanisms which physiologically protect the body from the development of autoimmune diseases and excessive tissue damage during inflammation by downregulating immune responses. This is frequently achieved by upregulation of immune checkpoints. Therefore, the blocking of immune checkpoint ligand-receptor interactions can reinstall the immune systems capability to fight cancer cells as shown for CTLA4 and PD-1 inhibitors in a clinical setting. Newly described checkpoint antigens are currently under investigation in cancer immunotherapy. Preclinical data emphasize the immune checkpoint axis TIGIT-PVR/PVRL2 as very promising target. This axis includes additional receptors such as DNAM-1, CD96, and CD112R. In this review, we discuss the recent findings of the relevance of this complex receptor ligand system in hematologic and solid cancers. Emphasis is also laid on the discussion of potential combinations with other immunotherapeutic approaches.
Assuntos
Nectinas/imunologia , Neoplasias/imunologia , Receptores Imunológicos/genética , Receptores Virais/genética , Antígenos CD/genética , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos T/genética , Antígenos de Diferenciação de Linfócitos T/imunologia , Antígeno CTLA-4/genética , Antígeno CTLA-4/imunologia , Humanos , Imunoterapia , Nectinas/genética , Neoplasias/genética , Neoplasias/terapia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/imunologia , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/imunologia , Receptores Imunológicos/imunologia , Receptores Virais/imunologiaRESUMO
BACKGROUND: Prostate cancer-related morbidity is associated with its preferential spread to the bone. Although the molecular interactions between the bone microenvironment and cancer cells have been researched extensively, the relevance of the microvascular properties of prostate cancer bone metastases remains largely unknown. Most preclinical studies focusing on microvascular analyses are based on heterotopic tumor implantation, whereas the impact of the microenvironment on site-specific growth behavior and angiogenesis is rarely addressed. METHODS: The microvascular changes associated with tumor growth in bone and soft tissue were characterized by implanting single cell suspensions of LnCap, Du145, and Pc3 cells into the femur (femur window) or striated muscle (dorsal skinfold chamber) of NSG mice. Tumor growth and the local microvasculature were analyzed for 21 days using intravital fluorescence microscopy. RESULTS: The results showed a higher engraftment of tumor cells in bone than in striated muscle associated with accelerated growth of LnCap cells and Pc3 cells. Permeability, blood flow, and tissue perfusion rates were greater in bone than in striated muscle. Du145 cells showed similar growth behavior in both tissues with similar vascular properties. The bone microenvironment facilitated tumor engraftment and growth. Increased microvascular density in striated muscle led to a higher tumor burden during early growth, whereas the increased perfusion promoted later prostate cancer growth in bone. CONCLUSIONS: Monitoring prostate cancer microcirculation in bone and soft tissue may be useful to evaluate the organ-specific efficacy of new treatments.
Assuntos
Neoplasias Ósseas/irrigação sanguínea , Neoplasias Ósseas/secundário , Fêmur/irrigação sanguínea , Músculo Estriado/irrigação sanguínea , Neovascularização Patológica , Neoplasias da Próstata/patologia , Animais , Linhagem Celular Tumoral , Humanos , Masculino , Camundongos , Modelos Animais , Transplante de Neoplasias , Microambiente TumoralRESUMO
OBJECTIVES: Leukaemia initiating cells reside within specialised niches in the bone marrow where they undergo complex interactions with different stromal cell types. The bone marrow niche is characterised by a low oxygen content resulting in high expression of hypoxia-inducible factor 1 α in leukaemic cells conferring a negative prognosis to patients with acute myeloid leukaemia (AML). METHODS AND RESULTS: In the current study, we investigated the impact of hypoxic vs. normoxic conditions on the sensitivity of AML cell lines and primary AML blasts to cytarabine. AML cells cultured under 6% oxygen were significantly more resistant against cytarabine compared to cells cultured under normoxic conditions in proliferation and colony-formation assays. Interestingly upon cultivation under hypoxia, the expression of the cytarabine-activating enzyme deoxycytidine kinase was downregulated in all analysed AML cell lines and primary AML samples representing a possible mechanism for resistance to chemotherapy. Furthermore, the downregulation of deoxycytidine kinase could be associated with hypoxia-inducible factor 1 α as treatment with its inhibitor BAY87-2243 hampered the downregulation of deoxycytidine kinase expression under hypoxic conditions. CONCLUSIONS: In conclusion, our data reveal that hypoxia-induced downregulation of deoxycytidine kinase represents one stroma-cell-independent mechanism of drug resistance to cytarabine in acute myeloid leukaemia.
Assuntos
Antimetabólitos Antineoplásicos/uso terapêutico , Citarabina/uso terapêutico , Desoxicitidina Quinase/metabolismo , Resistencia a Medicamentos Antineoplásicos , Hipóxia/metabolismo , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Antimetabólitos Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Citarabina/farmacologia , Desoxicitidina Quinase/genética , Regulação para Baixo , Expressão Gênica , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Leucemia Mieloide Aguda/genética , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismoRESUMO
BACKGROUND AND OBJECTIVES: Surgical interventions can alter the balance between pro- and anti-angiogenic growth factors and thereby modulate tumor growth. Since the microcirculatory properties of tumors underlie organ-specific differences, the microhemodynamic characteristics of bone metastasis have not yet been fully described. Angiogenesis inhibitors are increasingly being used to treat advanced stages of cancer. We hypothesized that the anti-angiogenic drug sunitinib abrogates alterations in microvascular properties following a minor surgical intervention in an in vivo model of secondary breast cancer growth in the bone. METHODS: Intravital microscopy was performed over 25 days using a xenograft model of breast cancer tumor growth in the bone to determine changes in microvascular properties during sunitinib treatment. Mastectomy was performed on day 5 to evaluate the effect of a minor surgical trauma on tumor growth and microvascular properties. RESULTS: Anti-angiogenic therapy resulted in reduced tumor growth, decreased vascular density, and increased vascular diameters. Blood flow velocity remained constant while microvascular permeability temporarily increased after the surgical intervention. CONCLUSIONS: Administration of sunitinib reduced tumor growth and altered microcirculatory properties in a time-dependent manner. The observed dramatic increase in microvascular permeability after the surgical intervention may have implications for local tumor growth, and metastatic dissemination. J. Surg. Oncol. 2016;113:515-521. © 2016 Wiley Periodicals, Inc.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/secundário , Neoplasias da Mama/patologia , Neoplasias da Mama/cirurgia , Indóis/uso terapêutico , Pirróis/uso terapêutico , Animais , Neoplasias Ósseas/irrigação sanguínea , Neoplasias da Mama/irrigação sanguínea , Feminino , Camundongos , Camundongos SCID , Microcirculação , Sunitinibe , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Acute myeloid leukemia (AML) represents a clonal disease of hematopoietic progenitors characterized by acquired heterogenous genetic changes that alter normal mechanisms of proliferation, self-renewal, and differentiation.(1) Although 40% to 45% of patients younger than 65 years of age can be cured with current therapies, only 10% of older patients reach long-term survival.(1) Because only very few novel AML drugs were approved in the past 2 decades, there is an urgent need to identify novel targets and therapeutic strategies to treat underserved AML patients. We report here that Axl, a member of the Tyro3, Axl, Mer receptor tyrosine kinase family,(2-4) represents an independent prognostic marker and therapeutic target in AML. AML cells induce expression and secretion of the Axl ligand growth arrest-specific gene 6 (Gas6) by bone marrow-derived stromal cells (BMDSCs). Gas6 in turn mediates proliferation, survival, and chemoresistance of Axl-expressing AML cells. This Gas6-Axl paracrine axis between AML cells and BMDSCs establishes a chemoprotective tumor cell niche that can be abrogated by Axl-targeting approaches. Axl inhibition is active in FLT3-mutated and FLT3 wild-type AML, improves clinically relevant end points, and its efficacy depends on presence of Gas6 and Axl. Axl inhibition alone or in combination with chemotherapy might represent a novel therapeutic avenue for AML.
Assuntos
Células da Medula Óssea/metabolismo , Leucemia Mieloide Aguda/metabolismo , Comunicação Parácrina/fisiologia , Proteínas Proto-Oncogênicas/metabolismo , Receptor Cross-Talk/fisiologia , Receptores Proteína Tirosina Quinases/metabolismo , Animais , Antineoplásicos/farmacocinética , Western Blotting , Ensaios Clínicos como Assunto , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Estimativa de Kaplan-Meier , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Masculino , Camundongos , Prognóstico , Reação em Cadeia da Polimerase em Tempo Real , Células Estromais/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Receptor Tirosina Quinase AxlRESUMO
Gremlin-1, an intrinsic antagonist of bone morphogenetic protein (BMP) signaling, has been implicated in the pathophysiology of pulmonary arterial hypertension (PAH). However, it is unknown whether gremlin-1 can be detected in the circulation of PAH patients and whether it is associated with patients' functional status and outcome. With a mean level of 242 ± 24 ng/ml, gremlin-1 levels of 31 PAH patients were significantly elevated compared to 151 ± 18 ng/ml in 15 age- and gender-matched healthy subject (p = 0.016). In PAH patients, increasing gremlin-1 levels correlated with N-terminal prohormone of brain natriuretic peptide levels (r = 0.608, p < 0.001) and inversely with the 6-minute walking distance (r = -0.412, p = 0.029). Furthermore, gremlin-1 significantly stratified survival in PAH patients (p = 0.015). Gremlin-1 may represent a new biomarker for PAH which can be linked directly to the underlying pathomechanism. Elevated levels of gremlin-1 are associated with patients' functional status and survival, thus gremlin-1 neutralization could represent a potential therapeutic strategy to increase BMPR2 signaling.
Assuntos
Hipertensão Pulmonar/sangue , Peptídeos e Proteínas de Sinalização Intercelular/sangue , Idoso , Biomarcadores/sangue , Proteínas Morfogenéticas Ósseas/metabolismo , Estudos de Casos e Controles , Teste de Esforço , Feminino , Taxa de Filtração Glomerular , Humanos , Hipertensão Pulmonar/fisiopatologia , Masculino , Pessoa de Meia-Idade , Peptídeo Natriurético Encefálico/sangue , Fragmentos de Peptídeos/sangue , Artéria Pulmonar , Transdução de Sinais , Taxa de Sobrevida , Caminhada/fisiologiaRESUMO
Since the approval of the first antiangiogenic agent bevacizumab, a neutralizing antibody against the vascular endothelial growth factor (VEGF), antiangiogenic therapies augmented the standard armamentarium of anticancer therapies and proved their clinical efficacy. Nevertheless, antiangiogenic strategies could not fulfill the expected hopes. In clinical routine, therapy responses to antiangiogenic therapies were mostly transient and most of the patients developed evasive resistance mechanisms during therapy. Further, no predictive biomarker for therapy response could be developed, hampering the clinical development of these agents and triggering skepticism. In the past years, knowledge on the biology of angiogenesis increased and the role of tumor hypoxia was better characterized and identified as the driver for angiogenic regulation mechanisms. Besides VEGF, new angiogenic and antiangiogenic factors were characterized and the process of endothelial cell migration, proliferation and vessel formation was better elucidated. Thus, a strong connection to neural development and axon guidance molecules like netrins, Slit proteins, semaphorins, ephrins and their cognate receptors UNC5, Robo1-4, neuropilin and EphB was identified. The aim of this review is to present the importance of these axon guidance molecules with special focus on Robo4 and semaphorins in tumor angiogenesis and to highlight their value as potential targets for new antiangiogenic therapies.
Assuntos
Inibidores da Angiogênese/farmacologia , Axônios/metabolismo , Semaforinas/fisiologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Neoplasias/irrigação sanguínea , Fatores de Crescimento Neural/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Netrina-1 , Receptores de Superfície Celular/fisiologia , Transdução de Sinais , Proteínas Supressoras de Tumor/fisiologia , Fator A de Crescimento do Endotélio Vascular/fisiologiaRESUMO
BACKGROUND: Tumors may develop resistance to specific angiogenic inhibitors via activation of alternative pathways. Therefore, multiple angiogenic pathways should be targeted to achieve significant angiogenic blockade. In this study we investigated the effects of a combined application of the angiogenic inhibitors endostatin and tumstatin in a model of human glioblastoma multiforme. RESULTS: Inhibitors released by stably transfected porcine aortic endothelial cells (PAE) showed anti-angiogenic activity in proliferation and wound-healing assays with endothelial cells (EC). Interestingly, combination of endostatin and tumstatin (ES + Tum) also reduced proliferation of glioma cells and additionally induced morphological changes and apoptosis in vitro. Microencapsulated PAE-cells producing these inhibitors were applied for local therapy in a subcutaneous glioblastoma model. When endostatin or tumstatin were applied separately, in vivo tumor growth was inhibited by 58% and 50%, respectively. Combined application of ES + Tum, in comparison, resulted in a significantly more pronounced inhibition of tumor growth (83%). cDNA microarrays of tumors treated with ES + Tum revealed an up-regulation of prolactin receptor (PRLR). ES + Tum-induced up-regulation of PRLR in glioma cells was also found in in vitro. Moreover, exogenous PRLR overexpression in vitro led to up-regulation of its ligand prolactin and increased proliferation suggesting a functional autocrine growth loop in these cells. CONCLUSION: Our data indicate that integrin-targeting factors endostatin and tumstatin act additively by inhibiting glioblastoma growth via reduction of vessel density but also directly by affecting proliferation and viability of tumor cells. Treatment with the ES + Tum-combination activates the PRLR pro-proliferative pathway in glioblastoma. Future work will show whether the prolactin signaling pathway represents an additional target to improve therapeutic strategies in this entity.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Glioblastoma/tratamento farmacológico , Integrinas/metabolismo , Receptores da Prolactina/metabolismo , Inibidores da Angiogênese/administração & dosagem , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Apoptose , Autoantígenos/administração & dosagem , Neoplasias Encefálicas/irrigação sanguínea , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Colágeno Tipo IV/administração & dosagem , Endostatinas/administração & dosagem , Glioblastoma/irrigação sanguínea , Glioblastoma/metabolismo , Glioblastoma/patologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Integrinas/antagonistas & inibidores , Camundongos , Camundongos SCID , Transdução de Sinais , Sus scrofa , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Introduction: Tumor-associated macrophages (TAMs) represent an important cell population within the tumor microenvironment, but little is known about the phenotype and function of these cells. The present study aims to characterize macrophages in high-grade serous ovarian cancer (HGSOC). Methods: Phenotype and expression of co-regulatory markers were assessed on TAMs derived from malignant ascites (MA) or peripheral blood (PB) by multiparametric flow cytometry. Samples were obtained from HGSOC patients (n=29) and healthy donors (HDs, n=16). Additional expression analysis was performed by RNAseq (n=192). Correlation with clinically relevant parameters was conducted and validated by a second patient cohort (n=517). Finally, the role of TIGIT in repolarization and phagocytosis was investigated in vitro. Results: Expression of the M2-associated receptors CD163, CD204, and CD206, as well as of the co-regulatory receptors TIGIT, CD226, TIM-3, and LAG-3 was significantly more frequent on macrophages in HGSOC than in HDs. CD39 and CD73 were broadly expressed on (mainly M2) macrophages, but without a clear clustering in HGSOC. CD163 mRNA levels were higher in TAMs from patients with residual tumor mass after surgery and associated with a shorter overall survival. In addition, TIGIT expression was associated with a higher tumor grading, indicating a prognostic relevance of M2 infiltration in HGSOC. TIGIT blockade significantly reduced the frequency of M2 macrophages. Moreover, combined blockade of TIGIT and CD47 significantly increased phagocytosis of ovarian cancer cells by TAMs in comparison to a single blockade of CD47. Conclusion: Combined blockade of TIGIT and CD47 represents a promising approach to enhance anti-CD47-facilitated phagocytosis.
Assuntos
Antígeno CD47 , Neoplasias Ovarianas , Humanos , Feminino , Antígeno CD47/genética , Antígeno CD47/metabolismo , Macrófagos Associados a Tumor/metabolismo , Fagocitose , Neoplasias Ovarianas/metabolismo , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Microambiente TumoralRESUMO
Chimeric antigen receptor T cells (CAR-T cells) have emerged as a powerful treatment option for individuals with B cell malignancies but have yet to achieve success in treating acute myeloid leukemia (AML) due to a lack of safe targets. Here we leveraged an atlas of publicly available RNA-sequencing data of over 500,000 single cells from 15 individuals with AML and tissue from 9 healthy individuals for prediction of target antigens that are expressed on malignant cells but lacking on healthy cells, including T cells. Aided by this high-resolution, single-cell expression approach, we computationally identify colony-stimulating factor 1 receptor and cluster of differentiation 86 as targets for CAR-T cell therapy in AML. Functional validation of these established CAR-T cells shows robust in vitro and in vivo efficacy in cell line- and human-derived AML models with minimal off-target toxicity toward relevant healthy human tissues. This provides a strong rationale for further clinical development.
Assuntos
Leucemia Mieloide Aguda , Transcriptoma , Humanos , Transcriptoma/genética , Linfócitos T , Imunoterapia Adotiva , Linhagem Celular , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Leucemia Mieloide Aguda/metabolismo , Linhagem Celular TumoralRESUMO
BACKGROUND: Leukemia-associated macrophages (LAMs) represent an important cell population within the tumor microenvironment, but little is known about the phenotype, function, and plasticity of these cells. The present study provides an extensive characterization of macrophages in patients with acute myeloid leukemia (AML). METHODS: The phenotype and expression of coregulatory markers were assessed on bone marrow (BM)-derived LAM populations, using multiparametric flow cytometry. BM and blood aspirates were obtained from patients with newly diagnosed acute myeloid leukemia (pAML, n=59), patients in long-term remission (lrAML, n=8), patients with relapsed acute myeloid leukemia (rAML, n=7) and monocyte-derived macrophages of the blood from healthy donors (HD, n=17). LAM subpopulations were correlated with clinical parameters. Using a blocking anti-T-cell immunoreceptor with Ig and ITIM domains (TIGIT) antibody or mouse IgG2α isotype control, we investigated polarization, secretion of cytokines, and phagocytosis on LAMs and healthy monocyte-derived macrophages in vitro. RESULTS: In pAML and rAML, M1 LAMs were reduced and the predominant macrophage population consisted of immunosuppressive M2 LAMs defined by expression of CD163, CD204, CD206, and CD86. M2 LAMs in active AML highly expressed inhibitory receptors such as TIGIT, T-cell immunoglobulin and mucin-domain containing-3 protein (TIM-3), and lymphocyte-activation gene 3 (LAG-3). High expression of CD163 was associated with a poor overall survival (OS). In addition, increased frequencies of TIGIT+ M2 LAMs were associated with an intermediate or adverse risk according to the European Leukemia Network criteria and the FLT3 ITD mutation. In vitro blockade of TIGIT shifted the polarization of primary LAMs or peripheral blood-derived M2 macrophages toward the M1 phenotype and increased secretion of M1-associated cytokines and chemokines. Moreover, the blockade of TIGIT augmented the anti-CD47-mediated phagocytosis of AML cell lines and primary AML cells. CONCLUSION: Our findings suggest that immunosuppressive TIGIT+ M2 LAMs can be redirected into an efficient effector population that may be of direct clinical relevance in the near future.
Assuntos
Leucemia Mieloide Aguda , Macrófagos , Animais , Camundongos , Fagocitose , Receptores Imunológicos/metabolismo , Fenótipo , Citocinas/metabolismo , Microambiente TumoralRESUMO
Phenotypic characterization of γδ T cells in the MALs (malignant ascites lymphocytes), TILs (tumor infiltrating lymphocytes), and PBLs (peripheral blood lymphocytes) of ovarian cancer (OvCA) patients is lacking. Therefore, we quantified γδ T cell prevalence in MAL, TIL, and PBL specimens from n = 18 OvCA patients and PBL from age-matched healthy donors (HD, n = 14). Multicolor flow cytometry was performed to evaluate the expression of inhibitory receptors (TIGIT, PD-1 and TIM-3), stimulatory receptors (Ox40), and purinergic ectoenzymes (CD39 and CD73) on γδ T cell subsets. We identified an abundant infiltration of Vδ1 T cells in the MALs and TILs. These cells varied in their differentiation: The majority of Vδ1 TILs displayed an effector memory (EM) phenotype, whereas Vδ1 MALs had a more mature phenotype of terminally differentiated effector memory cells (TEMRA) with high CD45RA expression. TIGIT and TIM-3 were abundantly expressed in both MALs and PBLs, whereas Vδ1 TILs exhibited the highest levels of PD-1, CD39, and Ox40. We also observed specific clusters on mature differentiation stages for the analyzed molecules. Regarding co-expression, Vδ1 TILs showed the highest levels of cells co-expressing TIGIT with PD-1 or CD39 compared to MALs and PBLs. In conclusion, the Vδ1 T cell population showed a high prevalence in the MALs and primary tumors of OvCA patients. Due to their (co-)expression of targetable immune receptors, in particular TIGIT with PD-1 and CD39 in TILs, Vδ1 T cell-based approaches combined with the inhibition of these targets might represent a promising strategy for OvCA.