RESUMO
The role played by the Notch pathway in cardiac progenitor cell biology remains to be elucidated. Delta-like ligand 4 (Dll4), the arterial-specific Notch ligand, is expressed by second heart field (SHF) progenitors at time-points that are crucial in SHF biology. Dll4-mediated Notch signaling is required for maintaining an adequate pool of SHF progenitors, such that Dll4 knockout results in a reduction in proliferation and an increase in apoptosis. A reduced SHF progenitor pool leads to an underdeveloped right ventricle (RV) and outflow tract (OFT). In its most severe form, there is severe RV hypoplasia and poorly developed OFT resulting in early embryonic lethality. In its milder form, the OFT is foreshortened and misaligned, resulting in a double outlet right ventricle. Dll4-mediated Notch signaling maintains Fgf8 expression by transcriptional regulation at the promoter level. Combined heterozygous knockout of Dll4 and Fgf8 demonstrates genetic synergy in OFT alignment. Exogenous supplemental Fgf8 rescues proliferation in Dll4 mutants in ex-vivo culture. Our results establish a novel role for Dll4-mediated Notch signaling in SHF biology. More broadly, our model provides a platform for understanding oligogenic inheritance that results in clinically relevant OFT malformations.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proliferação de Células , Fator 8 de Crescimento de Fibroblasto/biossíntese , Regulação da Expressão Gênica no Desenvolvimento , Ventrículos do Coração/embriologia , Receptores Notch/metabolismo , Transdução de Sinais , Células-Tronco/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas de Ligação ao Cálcio/genética , Fator 8 de Crescimento de Fibroblasto/genética , Camundongos , Camundongos Knockout , Receptores Notch/genéticaRESUMO
Introduction: Renal cell carcinoma (RCC) is primarily managed by surgery with the use of systemic targeted therapy in a metastatic setting. Newer targeted therapeutic options are evolving; Eph-ephrin is a potential new pathway. The therapeutic potential of targeting the EphB4-EphrinB2 pathway has been demonstrated in many solid tumors; however, its expression in RCC has only been evaluated in a few studies with limited cases. We herein determine the immunohistochemical expression of EphrinB2 in RCC. Methods: A tissue microarray comprising 110 cases of different histological subtypes of RCC and 10 normal kidney tissues were stained with monoclonal anti-EphrinB2 antibody (Abcam, AB201512). The tumor and endothelial cells expressing the EphrinB2 were examined and its expression was correlated with sex, histological subtypes, and tumor nodes metastasis (TNM) stage. Results: Twenty cases of urothelial carcinoma and two unsatisfactory conventional clear cell RCC cases were excluded, and EphrinB2 expression was interpreted in the remaining 88 tumors. EphrinB2 was expressed in 42 out of 88 tumors (47.7%) and was negative in the normal renal parenchyma. There was a statistically significant difference in the expression of EphrinB2 in males (55%) and females (32%). However, no such difference of expression was noted for the histological subtypes and the stages. Half (51%) of Stage 1 (n = 30) and Stage 2 (n = 11) tumors showed EphrinB2 positivity. Conclusions: EphrinB2 is expressed in approximately half of RCC cases. EphrinB2 expression in the early stage cancer might indicate its induction as an early event.
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The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the COVID-19 global pandemic, utilizes the host receptor angiotensin-converting enzyme 2 (ACE2) for viral entry. However, other host factors might also play important roles in SARS-CoV-2 infection, providing new directions for antiviral treatments. GRP78 is a stress-inducible chaperone important for entry and infectivity for many viruses. Recent molecular docking analyses revealed putative interaction between GRP78 and the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein (SARS-2-S). Here we report that GRP78 can form a complex with SARS-2-S and ACE2 on the surface and at the perinuclear region typical of the endoplasmic reticulum in VeroE6-ACE2 cells and that the substrate-binding domain of GRP78 is critical for this interaction. In vitro binding studies further confirmed that GRP78 can directly bind to the RBD of SARS-2-S and ACE2. To investigate the role of GRP78 in this complex, we knocked down GRP78 in VeroE6-ACE2 cells. Loss of GRP78 markedly reduced cell surface ACE2 expression and led to activation of markers of the unfolded protein response. Treatment of lung epithelial cells with a humanized monoclonal antibody (hMAb159) selected for its safe clinical profile in preclinical models depleted cell surface GRP78 and reduced cell surface ACE2 expression, as well as SARS-2-S-driven viral entry and SARS-CoV-2 infection in vitro. Our data suggest that GRP78 is an important host auxiliary factor for SARS-CoV-2 entry and infection and a potential target to combat this novel pathogen and other viruses that utilize GRP78 in combination therapy.
Assuntos
Enzima de Conversão de Angiotensina 2/genética , Proteínas de Choque Térmico/genética , Interações Hospedeiro-Patógeno/genética , SARS-CoV-2/efeitos dos fármacos , Glicoproteína da Espícula de Coronavírus/genética , Internalização do Vírus/efeitos dos fármacos , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Sítios de Ligação , Chlorocebus aethiops , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/virologia , Chaperona BiP do Retículo Endoplasmático , Regulação da Expressão Gênica , Proteínas de Choque Térmico/antagonistas & inibidores , Proteínas de Choque Térmico/metabolismo , Humanos , Mutação , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Transdução de Sinais , Glicoproteína da Espícula de Coronavírus/metabolismo , Resposta a Proteínas não Dobradas , Células VeroRESUMO
Pancreatic ductal adenocarcinoma (PDAC) remains a highly lethal disease in critical need of new therapeutic strategies. Here, we report that the stress-inducible 78-kDa glucose-regulated protein (GRP78/HSPA5), a key regulator of endoplasmic reticulum homeostasis and PI3K/AKT signaling, is overexpressed in the acini and PDAC of Pdx1-Cre;KrasG12D/+;p53f/+ (PKC) mice as early as 2 mo, suggesting that GRP78 could exert a protective effect on acinar cells under stress, as during PDAC development. The PKC pancreata bearing wild-type Grp78 showed detectable PDAC by 3 mo and rapid subsequent tumor growth. In contrast, the PKC pancreata bearing a Grp78f/+ allele (PKC78f/+ mice) expressing about 50% of GRP78 maintained normal sizes during the early months, with reduced proliferation and suppression of AKT, S6, ERK, and STAT3 activation. Acinar-to-ductal metaplasia (ADM) has been identified as a key tumor initiation mechanism of PDAC. Compared with PKC, the PKC78f/+ pancreata showed substantial reduction of ADM as well as pancreatic intraepithelial neoplasia-1 (PanIN-1), PanIN-2, and PanIN-3 and delayed onset of PDAC. ADM in response to transforming growth factor α was also suppressed in ex vivo cultures of acinar cell clusters isolated from mouse pancreas bearing targeted heterozygous knockout of Grp78 (c78f/+ ) and subjected to 3D culture in collagen. We further discovered that GRP78 haploinsufficiency in both the PKC78f/+ and c78f/+ pancreata leads to reduction of epidermal growth factor receptor, which is critical for ADM initiation. Collectively, our studies establish a role for GRP78 in ADM and PDAC development.
Assuntos
Carcinoma Ductal Pancreático/genética , Transdiferenciação Celular , Proteínas de Choque Térmico/genética , Pâncreas/patologia , Neoplasias Pancreáticas/genética , Células Acinares/metabolismo , Animais , Carcinogênese , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Chaperona BiP do Retículo Endoplasmático , Feminino , Haploinsuficiência , Proteínas de Choque Térmico/metabolismo , Masculino , Metaplasia , Camundongos , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Fator de Crescimento Transformador alfa/metabolismoRESUMO
PURPOSE: Conventional imaging cannot definitively detect nodal metastases of prostate cancer. We histologically validated C-acetate positron emission tomography/computerized tomography to identify nodal metastases, examining prostate cancer factors that influence detection rates. MATERIALS AND METHODS: Patients with C-acetate avid positron emission tomography/computerized tomography imaged pelvic/retroperitoneal lymph nodes underwent high extended robotic lymphadenectomy. A standardized mapping template comprising 8 predetermined anatomical regions was dissected during lymphadenectomy, allowing for matched, region based analysis and comparison of imaging and histological data. RESULTS: In 25 patients a total of 2,149 lymph nodes were excised (mean 86 per patient, range 27 to 136) and 528 (22%) harbored metastases (mean 21 positive nodes per patient, range 0 to 109). A total of 174 anatomical regions had matching imaging histological data. C-acetate positron emission tomography/computerized tomography accurately identified 48 node-positive regions and accurately ruled out 88 regions as metastasis-free. C-acetate sensitivity, specificity, and positive and negative predictive values were 67%, 84%, 74% and 79%, respectively. An increasing, histologically measured metastatic lesion size in long axis diameter of 5 or less, 6 to 10, 11 to 15, 16 to 20 and 21 mm or greater correlated with improved C-acetate detection rates of 45%, 62%, 81%, 89% and 100%, respectively. Each standard uptake value unit increase correlated with a 1.9 mm increase in nodal long axis diameter (p <0.001) and a 1.2 mm increase in short axis diameter (p <0.001). Positive C-acetate positron emission tomography/computerized tomography findings correlated with histological lymph node size (long axis diameter 12 mm and short axis diameter 6 mm), metastatic lesion size (long axis diameter 11 mm and short axis diameter 6 mm) and extranodal extension (positive 88% vs false-negative 58%, p = 0.005). CONCLUSIONS: C-acetate positron emission tomography/computerized tomography can identify prostate cancer metastatic nodal disease. However, it underestimates the true cephalad extent of nodal involvement, performing better in the pelvis than in the retroperitoneum. Standard uptake value, histological nodal size, intranodal metastasis size and extranodal extension correlate with cancer bearing nodes.
Assuntos
Radioisótopos de Carbono/administração & dosagem , Metástase Linfática/diagnóstico por imagem , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada/métodos , Neoplasias da Próstata/patologia , Compostos Radiofarmacêuticos/administração & dosagem , Idoso , Reações Falso-Negativas , Humanos , Excisão de Linfonodo/métodos , Linfonodos/diagnóstico por imagem , Linfonodos/patologia , Linfonodos/cirurgia , Metástase Linfática/patologia , Masculino , Pessoa de Meia-Idade , Pelve/diagnóstico por imagem , Estudos Prospectivos , Próstata/patologia , Neoplasias da Próstata/diagnóstico por imagem , Espaço Retroperitoneal/diagnóstico por imagem , Procedimentos Cirúrgicos Robóticos/métodos , Sensibilidade e EspecificidadeRESUMO
OBJECTIVES: To describe the technique of robot-assisted high-extended salvage retroperitoneal and pelvic lymphadenectomy (sRPLND+PLND) for 'node-only' recurrent prostate cancer. PATIENTS AND METHODS: In all, 10 patients underwent robot-assisted sRPLND+PLND (09/2015-03/2016) for 'node-only' recurrent prostate cancer, as identified by 11 C-acetate positron emission tomography/computed tomography imaging. Our anatomical template extends from bilateral renal artery/vein cranially up to Cloquet's node caudally, completely excising lymphatic-fatty tissue from aorto-caval and iliac vascular trees; RPLND precedes PLND. Meticulous node-mapping assessed nodes at four prospectively assigned anatomical zones. RESULTS: The median operative time was 4.8 h, estimated blood loss 100 mL and hospital stay 1 day. No patient had an intraoperative complication, open conversion or blood transfusion. Three patients had spontaneously resolving Clavien-Dindo grade II postoperative complications. The mean (range) number of nodes excised per patient was 83 (41-132) and mean (range) number of positive nodes per patient was 23 (0-109). Seven patients (70%) had positive nodes on final pathology. Node-positive rates per anatomical level I, II, III and IV were 28%, 32%, 33% and 33%, respectively. In patients with positive nodes, the median PSA level had decreased by 83% at the 2-month follow-up. CONCLUSION: The initial series of robot-assisted sRPLND+PLND is presented, wherein we duplicate open surgery with superior nodal counts and decreased morbidity. Robot-assisted technical details for an anatomical LND template up to the renal vessels are presented. Longer follow-up is necessary to assess oncological outcomes.
Assuntos
Excisão de Linfonodo/métodos , Linfonodos/cirurgia , Neoplasias da Próstata/patologia , Neoplasias Retroperitoneais/cirurgia , Procedimentos Cirúrgicos Robóticos/métodos , Terapia de Salvação/métodos , Idoso , Humanos , Linfonodos/diagnóstico por imagem , Linfonodos/patologia , Masculino , Pessoa de Meia-Idade , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Recidiva , Neoplasias Retroperitoneais/diagnóstico por imagem , Neoplasias Retroperitoneais/patologiaRESUMO
Delta-like ligand 4 (Dll4) expressed in tumor cells plays a key role to promote tumor growth of numerous cancer types. Based on a novel antihuman Dll4 monoclonal antibody (61B), we developed a (64)Cu-labeled probe for positron emission tomography (PET) imaging of tumor Dll4 expression. In this study, 61B was conjugated with the (64)Cu-chelator DOTA through lysine on the antibody. Human IgG (hIgG)-DOTA, which did not bind to Dll4, was also prepared as a control. The Dll4 binding activity of the probes was evaluated through the bead-based binding assay with Dll4-alkaline phosphatase. The resulting PET probes were evaluated in U87MG glioblastoma and HT29 colorectal cancer xenografts in athymic nude mice. Our results demonstrated that the 61B-DOTA retained (77.2 ± 3.7) % Dll4 binding activity of the unmodified 61B, which is significantly higher than that of hIgG-DOTA (0.06 ± 0.03) %. Confocal microscopy analysis confirmed that 61B-Cy5.5, but not IgG-Cy5.5, predominantly located within the U87MG and HT29 cells cytoplasm. U87MG cells showed higher 61B-Cy5.5 binding as compared to HT29 cells. In U87MG xenografts, 61B-DOTA-(64)Cu demonstrated remarkable tumor accumulation (10.5 ± 1.7 and 10.2 ± 1.2%ID/g at 24 and 48 h postinjection, respectively). In HT29 xenografts, tumor accumulation of 61B-DOTA-(64)Cu was significantly lower than that of U87MG (7.3 ± 1.3 and 6.6 ± 1.3%ID/g at 24 and 48 h postinjection, respectively). The tumor accumulation of 61B-DOTA-(64)Cu was significantly higher than that of hIgG-DOTA-(64)Cu in both xenografts models. Immunofluorescence staining of the tumor tissues further confirmed that tumor accumulation of 61B-Cy5.5 was correlated well with in vivo PET imaging data using 61B-DOTA-(64)Cu. In conclusion, 61B-DOTA-(64)Cu PET probe was successfully synthesized and demonstrated prominent tumor uptake by targeting Dll4. 61B-DOTA-(64)Cu has great potential to be used for noninvasive Dll4 imaging, which could be valuable for tumor detection, Dll4 expression level evaluation, and Dll4-based treatment monitoring.
Assuntos
Neoplasias Colorretais/metabolismo , Glioblastoma/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Proteínas Adaptadoras de Transdução de Sinal , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Proteínas de Ligação ao Cálcio , Linhagem Celular Tumoral/transplante , Radioisótopos de Cobre/uso terapêutico , Feminino , Células HT29/transplante , Compostos Heterocíclicos com 1 Anel/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Camundongos , Camundongos Nus , Microscopia Confocal , Transplante de Neoplasias , Tomografia por Emissão de PósitronsRESUMO
Solid malignancies are often characterized by overexpression of various receptor tyrosine kinases (RTKs) against which many targeted therapies are currently in use and in active development. EPHB4 has recently emerged as a frequently overexpressed RTK in many types of cancer. Here, we demonstrate expression patterns of EPHB4 in two solid malignancies: squamous cell carcinoma of the head and neck (HNSCC) and renal cell carcinoma (RCC), by immunohistochemical analysis. We demonstrate the first association between EPHB4 expression and progression of HNSCC from normal tissue to dysplasia and to cancer. Interestingly, most RCC subtypes exhibited expression patterns that were opposite from that found in HNSCC, possibly owing to their unique biology and high degree of organ and tumor vasculature. Taken together, these results suggest a possible role for EPHB4 as a therapeutic target in these malignancies.
Assuntos
Carcinoma de Células Renais/metabolismo , Carcinoma de Células Escamosas/metabolismo , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias Renais/metabolismo , Receptor EphB4/metabolismo , Carcinoma de Células Renais/patologia , Carcinoma de Células Escamosas/patologia , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Neoplasias Renais/patologia , Receptor EphB4/genéticaRESUMO
Accumulating experimental evidence indicates that overexpression of the oncogenic receptor tyrosine kinase, Axl, plays a key role in the tumorigenesis and metastasis of various types of cancer. The objective of this study is to design a novel imaging probe based on the monoclonal antibody, h173, for microPET imaging of Axl expression in human lung cancer. A bifunctional chelator, DOTA, was conjugated to h173, followed by radiolabeling with (64)Cu. The binding of DOTA-h173 to the Axl receptor was first evaluated by a cell uptake assay and flow cytometry analysis using human lung cancer cell lines. The probe (64)Cu-DOTA-h173 was further evaluated by microPET imaging, and ex vivo histology studies in the Axl-positive A549 tumors. In vitro cellular study showed that Axl probe, (64)Cu-DOTA-h173, was highly immuno-reactive with A549 cells. Western blot analysis confirmed that Axl is highly expressed in the A549 cell line. For microPET imaging, the A549 xenografts demonstrated a significantly higher (64)Cu-DOTA-h173 uptake compared to the NCI-H249 xenograft (a negative control model). Furthermore, (64)Cu-DOTA-h173 uptake in A549 is significantly higher than that of (64)Cu-DOTA-hIgG. Immuno-fluorescence staining was consistent with the in vivo micro-PET imaging results. In conclusion, (64)Cu-DOTA-h173 could be potentially used as a probe for noninvasive imaging of Axl expression, which could collect important information regarding tumor response to Axl-targeted therapeutic interventions.
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Anticorpos Monoclonais Humanizados , Carcinoma Pulmonar de Células não Pequenas/diagnóstico por imagem , Radioisótopos de Cobre , Desenho de Fármacos , Tomografia por Emissão de Pósitrons/métodos , Proteínas Proto-Oncogênicas/imunologia , Compostos Radiofarmacêuticos , Receptores Proteína Tirosina Quinases/imunologia , Animais , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/farmacocinética , Western Blotting , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Radioisótopos de Cobre/farmacocinética , Citometria de Fluxo , Imunofluorescência , Humanos , Neoplasias Pulmonares/diagnóstico por imagem , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Nus , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Compostos Radiofarmacêuticos/farmacocinética , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Distribuição Tecidual , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto , Receptor Tirosina Quinase AxlRESUMO
OBJECTIVE: Patients with relapsed or metastatic head and neck squamous cell carcinoma (HNSCC) after primary local therapy have low response rates with cetuximab, systemic chemotherapy or check point inhibitor therapy. Novel combination therapies with the potential to improve outcomes for patients with HNSCC is an area of high unmet need. METHODS: This is a phase II single-arm clinical trial of locally advanced or metastatic HNSCC patients treated with a combination of soluble EphB4-human serum albumin (sEphB4-HSA) fusion protein and pembrolizumab after platinum-based chemotherapy with up to 2 prior lines of treatment. The primary endpoints were safety and tolerability and the primary efficacy endpoint was overall response rate (ORR). Secondary endpoints included progression free survival (PFS) and overall survival (OS). HPV status and EphrinB2 expression were evaluated for outcome. RESULTS: Twenty-five patients were enrolled. Median follow up was 40.4 months (range 9.8 - 40.4). There were 6 responders (ORR 24%). There were 5 responders in the 11 HPV-negative and EphrinB2 positive patients, (ORR 45%) with 2 of these patients achieving a complete response (CR). The median PFS in HPV-negative/EphrinB2 positive patients was 3.2 months (95% CI 1.1, 7.3). Median OS in HPV-negative/EphrinB2 positive patients was 10.9 months (95% CI 2.0, 13.7). Hypertension, transaminitis and fatigue were the most common toxicities. DISCUSSION: The combination of sEphB4-HSA and pembrolizumab has a favorable toxicity profile and favorable activity particularly among HPV-negative EphrinB2 positive patients with HNSCC.
Assuntos
Anticorpos Monoclonais Humanizados , Efrina-B2 , Neoplasias de Cabeça e Pescoço , Receptor EphB4 , Carcinoma de Células Escamosas de Cabeça e Pescoço , Humanos , Anticorpos Monoclonais Humanizados/uso terapêutico , Feminino , Masculino , Pessoa de Meia-Idade , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Idoso , Efrina-B2/metabolismo , Adulto , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Receptor EphB4/metabolismo , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Infecções por Papillomavirus/virologia , Resultado do Tratamento , Proteínas Recombinantes de Fusão/uso terapêutico , Idoso de 80 Anos ou maisRESUMO
BACKGROUND: Malignant pleural mesothelioma (MPM) often develops decades following exposure to asbestos. Current best therapy produces a response in only half of patients, and the median survival with this therapy remains under a year. A search for novel targets and therapeutics is underway, and recently identified targets include VEGF, Notch, and EphB4-Ephrin-B2. Each of these targets has dual activity, promoting tumor cell growth as well as tumor angiogenesis. METHODS: We investigated EphB4 expression in 39 human mesothelioma tissues by immunohistochemistry. Xenograft tumors established with human mesothelioma cells were treated with an EphB4 inhibitor (monomeric soluble EphB4 fused to human serum albumin, or sEphB4-HSA). The combinatorial effect of sEphB4-HSA and biologic agent was also studied. RESULTS: EphB4 was overexpressed in 72% of mesothelioma tissues evaluated, with 85% of epithelioid and 38% of sarcomatoid subtypes demonstrating overexpression. The EphB4 inhibitor sEphB4-HSA was highly active as a single agent to inhibit tumor growth, accompanied by tumor cell apoptosis and inhibition of PI3K and Src signaling. Combination of sEphB4-HSA and the anti-VEGF antibody (Bevacizumab) was superior to each agent alone and led to complete tumor regression. CONCLUSION: EphB4 is a potential therapeutic target in mesothelioma. Clinical investigation of sEphB4-HSA as a single agent and in combination with VEGF inhibitors is warranted.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Mesotelioma/metabolismo , Neoplasias Pleurais/metabolismo , Receptor EphB4/metabolismo , Animais , Anticorpos Monoclonais Humanizados/administração & dosagem , Bevacizumab , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos , Imunofluorescência , Humanos , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Receptor EphB4/administração & dosagem , Albumina Sérica/administração & dosagem , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Accumulating evidence suggests that overexpression of the tyrosine kinase receptor EphB4, a mediator of vascular development, is a novel target for tumor diagnosis, prognosis and therapy. Noninvasive imaging of EphB4 expression could therefore be valuable for evaluating disease course and therapeutic efficacy at the earliest stages of anti-EphB4 treatment. In this study, we systematically investigated the use of anti-EphB4 antibody h131 (150 kDa) and its fragments (h131-F(ab')2, 110 kDa; h131-Fab, 50 kDa) for near-infrared fluorescence (NIRF) imaging of EphB4 expression in vivo. h131-F(ab')2 and h131-Fab were produced through pepsin and papain digestion of h131 respectively, whose purity was confirmed by FPLC and SDS-PAGE. After conjugation with Cy5.5, in vivo characteristics of h131, h131-F(ab')2 and h131-Fab were evaluated in EphB4-positive HT29 tumor model. Although h131-Cy5.5 demonstrated highest tumor uptake among these probes, its optimal tumor uptake level was obtained at 2 days post injection (p.i.). For h131-Fab-Cy5.5, maximum tumor uptake was achieved at 4 h p.i. However, no significant difference was observed between h131-Fab-Cy5.5 and hIgG-Fab-Cy5.5, indicating the tumor accumulation was mainly caused by passive targeting. In contrast, h131-F(ab')2-Cy5.5 demonstrated prominent tumor uptake at 6 h p.i. The target specificity was confirmed by hIgG-F(ab')2-Cy5.5 control and immunofluorescent staining. Collectively, h131-F(ab')2 exhibited prominent and specific tumor uptake at early time points, which suggests it is a promising agent for EphB4-targeted imaging.
Assuntos
Anticorpos Monoclonais/imunologia , Neoplasias/diagnóstico , Neoplasias/imunologia , Receptor EphB4/imunologia , Linhagem Celular Tumoral , Diagnóstico por Imagem/métodos , Células HT29 , Humanos , Distribuição Tecidual/imunologiaRESUMO
Accumulating evidence suggests that EphB4 plays key roles in cancer progression in numerous cancer types. In fact, therapies focusing on EphB4 have become potentially important components of various cancer treatment strategies. However, tumor sensitivity to EphB4 suppression may not be uniform for different cancers. In this study, we developed near-infrared fluorescence (NIRF) probes for EphB4 targeted imaging, based on EphB4-specific humanized monoclonal antibody hAb47. NIRF dye Cy5.5 was introduced to hAb47 either through the reaction with amino groups (named hAb47-Cy5.5) or sulfhydryl groups (named hAb47-Cy5.5-Mal). The resulting probes were evaluated in both HT-29 xenograft and the mAb131 (anti-EphB4) treated models. Although these methods lead to modifications of both the heavy chain and light chain of the antibody, the majority of the EphB4 binding affinity was maintained (81.62 ± 2.08% for hAb47-Cy5.5 and 77.14 ± 2.46% for hAb47-Cy5.5-Mal, respectively). hAb47-Cy5.5 was then chosen for in vivo NIRF imaging of EphB4 expression. In HT29 colorectal tumor xenografts, hAb47-Cy5.5 demonstrated significantly higher tumor uptake compared with that of the hIgG-Cy5.5 control, which was further confirmed by immunofluorescent staining. Moreover, hAb47-Cy5.5 successfully imaged the decreased EphB4 expression (confirmed by Western blot) in EphB4-targeted immunotherapy using another EphB4-specific antibody, mAb131. Collectively, hAb47-Cy5.5 could be used as a specific NIRF contrast agent for noninvasive imaging of EphB4 expression, which may predict whether an individual tumor would likely respond to EphB4 targeted interventions, as well as monitor the therapeutic response.
Assuntos
Carbocianinas/farmacocinética , Diagnóstico por Imagem/métodos , Corantes Fluorescentes/farmacocinética , Neoplasias/diagnóstico , Neoplasias/metabolismo , Receptor EphB4/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados/metabolismo , Linhagem Celular Tumoral , Monitoramento Ambiental/métodos , Feminino , Células HT29 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias/terapia , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Patients with metastatic urothelial carcinoma have poor prognosis after failure of standard first-line chemotherapy. Immune check point programmed death 1-programmed death ligand 1 antibodies have low response rates and thus there exists a major unmet need. MATERIALS AND METHODS: In this phase II trial, patients with metastatic urothelial carcinoma that recurred or progressed after platinum-based chemotherapy received soluble EphB4-human serum albumin (sEphB4-HSA) in combination with pembrolizumab. The primary end points were tolerability and overall survival (OS). The secondary end points were progression-free survival (PFS), objective response rate (ORR), duration of response, and toxicity. The expression of sEphB4-HSA target EphrinB2 was correlated with outcomes. RESULTS: Seventy patients were enrolled. The median follow up was 22.9 months (range, 1.3-54.7). The regimen had acceptable toxicity. In the intent-to-treat analysis (N = 70), the median OS was 14.6 months (95% CI, 9.2 to 21.5). Twenty-six (37%) patients had an objective response (95% CI, 26 to 48). The median PFS was 4.1 (95% CI, 1.5 to 5.7) months. Forty-six (66%) patients expressed EphrinB2, and among them, the median OS was 21.5 months (95% CI, 12.4 to not reached), the ORR was 52% (95% CI, 37 to 67), including a complete response rate of 24% (11 of 46; 95% CI, 12 to 36). The median PFS was 5.7 (95% CI, 2.7 to 27.9) months. Response was maintained at 6, 12, and 24 months in 88%, 74%, and 69% of the patients, respectively. CONCLUSION: The combination of sEphB4-HSA and pembrolizumab appears synergistic with improved OS and ORR compared with historical data for programmed death 1/programmed death ligand 1 monotherapy.
Assuntos
Anticorpos Monoclonais Humanizados , Carcinoma de Células de Transição , Efrina-B2 , Neoplasias da Bexiga Urinária , Humanos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma de Células de Transição/tratamento farmacológico , Efrina-B2/antagonistas & inibidores , Recidiva Local de Neoplasia/tratamento farmacológico , Neoplasias da Bexiga Urinária/tratamento farmacológico , Anticorpos Monoclonais Humanizados/uso terapêuticoRESUMO
Notch pathway regulates vessel development and maturation. Dll4, a high-affinity ligand for Notch, is expressed predominantly in the arterial endothelium and is induced by hypoxia among other factors. Inhibition of Dll4 has paradoxical effects of reducing the maturation and perfusion in newly forming vessels while increasing the density of vessels. We hypothesized that partial and/or intermittent inhibition of Dll4 may lead to increased vascular response and still allow vascular maturation to occur. Thus tissue perfusion can be restored rapidly, allowing quicker recovery from ischemia or tissue injury. Our studies in two different models (hindlimb ischemia and skin flap) show that inhibition of Dll4 at low dose allows faster recovery from vascular and tissue injury. This opens a new possibility for Dll4 blockade's therapeutic application in promoting recovery from vascular injury and restoring blood supply to ischemic tissues.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Isquemia/tratamento farmacológico , Proteínas de Membrana/antagonistas & inibidores , Receptores Notch/antagonistas & inibidores , Proteínas Recombinantes de Fusão/uso terapêutico , Animais , Vasos Sanguíneos/efeitos dos fármacos , Modelos Animais de Doenças , Imunofluorescência , Retalhos de Tecido Biológico/irrigação sanguínea , Membro Posterior/irrigação sanguínea , Camundongos , Camundongos Mutantes , Reperfusão , Transdução de Sinais/efeitos dos fármacos , Pele/irrigação sanguínea , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/biossínteseRESUMO
Axl is an oncogenic receptor tyrosine kinase that plays multiple roles in tumorigenesis and metastasis of many cancers. This study is the first to demonstrate that Axl is induced in Kaposi sarcoma and Kaposi sarcoma herpesvirus (KSHV) transformed endothelial cells. Conditionally, expression of one KSHV latency protein vFLIP induces Axl expression in endothelial cells. This induction can be blocked by nuclear factor-kappaB inhibitor, consistent with the known vFLIP mechanism of action. KS cell lines lacking KSHV also have elevated Axl expression, which probably resulted from hypomethylation of AXL promoter. Axl activation activates downstream phosphoinositol-3 kinase signaling, and Axl knockdown by siRNA impairs phosphoinositol-3 kinase signaling. Furthermore, Axl knockdown inhibits KS cell growth and invasion. To explore the potential for translation of these findings, we generated monoclonal antibodies to block the biologic functions of Axl. MAb173, which induces receptor degradation, showed activity in vitro to inhibit KS cell invasion. Moreover, in vivo xenograft studies with KS cells with or without KSHV infection showed that MAb173 reduced tumor growth, increased tumor cell apoptosis, and markedly decreased Axl protein level in tumors. Axl thus has a potential role in KS pathogenesis and is a candidate for prognostic and therapeutic investigations.
Assuntos
Transformação Celular Viral/genética , Regulação Neoplásica da Expressão Gênica , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Sarcoma de Kaposi/metabolismo , Transdução de Sinais/fisiologia , Animais , Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Western Blotting , Metilação de DNA , Células Endoteliais/virologia , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Dosagem de Genes , Herpesvirus Humano 8 , Humanos , Imunoprecipitação , Camundongos , Camundongos Endogâmicos BALB C , Invasividade Neoplásica/genética , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas/imunologia , Proteínas Proto-Oncogênicas/metabolismo , RNA Interferente Pequeno , Receptores Proteína Tirosina Quinases/imunologia , Receptores Proteína Tirosina Quinases/metabolismo , Sarcoma de Kaposi/genética , Sarcoma de Kaposi/virologia , Transfecção , Proteínas Virais/genética , Proteínas Virais/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Receptor Tirosina Quinase AxlRESUMO
Kaposi sarcoma-associated herpesvirus (KSHV) infection is essential to the development of Kaposi sarcoma (KS). Notch signaling is also known to play a pivotal role in KS cell survival and lytic phase entrance of KSHV. In the current study, we sought to determine whether KSHV regulates Notch components. KSHV-infected lymphatic endothelial cells showed induction of receptors Notch3 and Notch4, Notch ligands Dll4 and Jagged1, and activated Notch receptors in contrast to uninfected lymphatic endothelial cells. In addition, KSHV induced the expression of endothelial precursor cell marker (CD133) and mural cell markers (calponin, desmin, and smooth muscle alpha actin), suggesting dedifferentiation and trans-differentiation. Overexpression of latency proteins (LANA, vFLIP) and lytic phase proteins (RTA, vGPCR, viral interleukin-6) further supported the direct regulatory capacity of KSHV viral proteins to induce Notch receptors (Notch2, Notch3), ligands (Dll1, Dll4, Jagged1), downstream targets (Hey, Hes), and endothelial precursor CD133. Targeting Notch pathway with gamma-secretase inhibitor and a decoy protein in the form of soluble Dll4 inhibited growth of KSHV-transformed endothelial cell line. Soluble Dll4 was also highly active in vivo against KS tumor xenograft. It inhibited tumor cell growth, induced tumor cell death, and reduced vessel perfusion. Soluble Dll4 is thus a candidate for clinical investigation.
Assuntos
Células Endoteliais , Infecções por Herpesviridae/metabolismo , Herpesvirus Humano 8 , Receptores Notch/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Sobrevivência Celular/fisiologia , Transformação Celular Viral , Células Cultivadas , Células Endoteliais/citologia , Células Endoteliais/fisiologia , Células Endoteliais/virologia , Regulação Viral da Expressão Gênica , Infecções por Herpesviridae/patologia , Infecções por Herpesviridae/fisiopatologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteína Jagged-1 , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA Interferente Pequeno , Receptor Notch1/genética , Receptor Notch1/metabolismo , Receptor Notch2/genética , Receptor Notch2/metabolismo , Receptor Notch3 , Receptor Notch4 , Receptores Notch/genética , Proteínas Serrate-Jagged , Transfecção , Transplante Heterólogo , Artérias Umbilicais/citologia , Veias Umbilicais/citologia , Latência Viral/fisiologiaRESUMO
The role of surgical experience and its impact on the survival requires further investigation. A cohort of patients undergoing radical cystectomy or anterior pelvic exenteration for localized bladder cancer between 2006 and 2013 at 1143 facilities across the United States was identified using the National Cancer Database and analyzed. Using overall survival (OS) as the primary outcome, the relationship between facility annual caseload (FAC) and facility annual surgical caseload (FASC) for those undergoing curative surgery was examined. Four volume groups (VG) depending on caseload using both FAC and FASC were defined. These included VG1: below 50th percentile, VG2: 50th−74th percentile, VG3: 75th−89th percentile, and VG4: 90th and above. Between 2006 and 2013, 27,272 patients underwent surgery for localized bladder cancer. The median OS was 59.66 months (95% CI: 57.79−61.77). OS improved significantly as caseload increased. The unadjusted median OS difference between VG1 and VG4 was 15.35 months (64.3 vs. 48.95 months, HR 1.19 95% CI: 1.13−1.25, p < 0.001) for FAC. This figure was 19.84 months (66.89 vs. 47.05 months, HR 1.25 95% CI: 1.18−1.32, p < 0.0001) for FASC. This analysis revealed a significant and clinically important survival advantage for curative bladder cancer surgery at highly experienced centers.
RESUMO
The recent discovery that GRP78/BiP, a typical endoplasmic reticulum (ER) lumenal chaperone, can be expressed on the cell surface, interacting with an increasing repertoire of surface proteins and acting as receptor in signaling pathways, represents a paradigm shift in its biological function. However, the mechanism of GRP78 trafficking from the ER to the cell surface is not well understood. Using a combination of cellular, biochemical, and mutational approaches, we tested multiple hypotheses. Here we report that ER stress actively promotes GRP78 localization on the cell surface, whereas ectopic expression of GRP78 is also able to cause cell surface relocation in the absence of ER stress. Moreover, deletion of the C-terminal ER retention motif in GRP78 alters its cell surface presentation in a dose-dependent manner; however, mutation of the putative O-linked glycosylation site Thr(648) of human GRP78 is without effect. We also identified the exposure of multiple domains of GRP78 on the cell surface and determined that binding of extracellular GRP78 to the cell surface is unlikely. A new topology model for cell surface GRP78 is presented.
Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Chaperona BiP do Retículo Endoplasmático , Epitopos , Citometria de Fluxo , Glicosilação , Proteínas de Choque Térmico/genética , Humanos , Mutação , PlasmídeosRESUMO
EphB4 receptor tyrosine kinase and its cognate ligand EphrinB2 regulate induction and maturation of newly forming vessels. Inhibition of their interaction arrests angiogenesis, vessel maturation, and pericyte recruitment. In addition, EphB4 is expressed in the vast majority of epithelial cancers and provides a survival advantage to most. Here, we describe two anti-EphB4 monoclonal antibodies that inhibit tumor angiogenesis and tumor growth by two distinct pathways. MAb131 binds to fibronectin-like domain 1 and induces degradation of human EphB4, but not murine EphB4. MAb131 inhibits human endothelial tube formation in vitro and growth of human tumors expressing EphB4 in vivo. In contrast, MAb47 targets fibronectin-like domain 2 of both human and murine EphB4 and does not alter EphB4 receptor levels, but inhibits angiogenesis and growth of both EphB4-positive and EphB4-negative tumors in a mouse s.c. xenograft model. Combination of MAb47 and bevacizumab enhances the antitumor activity and induces tumor regression. Indeed, humanized antibodies hAb47 and hAb131 showed similar affinity for EphB4 and retained efficacy in the inhibition of primary tumor development and experimental metastasis.