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
Optimal cytoreduction for ovarian cancer is often challenging because of aggressive tumor biology and advanced stage. It is a critical issue since the extent of residual disease after surgery is the key predictor of ovarian cancer patient survival. For a limited number of cancers, fluorescence-guided surgery has emerged as an effective aid for tumor delineation and effective cytoreduction. The intravenously administered fluorescent agent, most commonly indocyanine green (ICG), accumulates preferentially in tumors, which are visualized under a fluorescent light source to aid surgery. Insufficient tumor specificity has limited the broad application of these agents in surgical oncology including for ovarian cancer. In this study, we developed a novel tumor-selective fluorescent agent by chemically linking ICG to mouse monoclonal antibody 10D7 that specifically recognizes an ovarian cancer-enriched cell surface receptor, CUB-domain-containing protein 1 (CDCP1). 10D7ICG has high affinity for purified recombinant CDCP1 and CDCP1 that is located on the surface of ovarian cancer cells in vitro and in vivo. Our results show that intravenously administered 10D7ICG accumulates preferentially in ovarian cancer, permitting visualization of xenograft tumors in mice. The data suggest CDCP1 as a rational target for tumor-specific fluorescence-guided surgery for ovarian cancer.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Moléculas de Adesão Celular/antagonistas & inibidores , Corantes Fluorescentes/administração & dosagem , Imagem Óptica/métodos , Neoplasias Ovarianas/diagnóstico , Animais , Anticorpos Monoclonais/química , Antígenos de Neoplasias , Linhagem Celular Tumoral , Feminino , Corantes Fluorescentes/química , Humanos , Verde de Indocianina/administração & dosagem , Verde de Indocianina/química , Injeções Intravenosas , Camundongos , Neoplasias Ovarianas/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The cellular receptor CUB domain containing protein 1 (CDCP1) is commonly elevated and functionally important in a range of cancers. CDCP1 is cleaved by serine proteases at adjacent sites, arginine 368 (R368) and lysine 369 (K369), which induces cell migration in vitro and metastasis in vivo. We demonstrate that membrane localization of serine protease activity increases efficacy of cleavage of CDCP1, and that both secreted and membrane anchored serine proteases can have distinct preferences for cleaving at CDCP1-R368 and CDCP1-K369. Approaches that disrupt membrane localization of CDCP1 cleaving serine proteases may interfere with the cancer promoting effects of CDCP1 proteolysis.
Assuntos
Antígenos CD/metabolismo , Moléculas de Adesão Celular/metabolismo , Membrana Celular/enzimologia , Neoplasias Renais/metabolismo , Proteínas de Neoplasias/metabolismo , Receptores Ativados por Proteinase/metabolismo , Serina Proteases/metabolismo , Antígenos de Neoplasias , Linhagem Celular Tumoral , Movimento Celular , Humanos , Neoplasias Renais/patologia , ProteóliseRESUMO
Rationale: An antibody-drug conjugate (ADC) is a targeted therapy consisting of a cytotoxic payload that is linked to an antibody which targets a protein enriched on malignant cells. Multiple ADCs are currently used clinically as anti-cancer agents significantly improving patient survival. Herein, we evaluated the rationale of targeting the cell surface oncoreceptor CUB domain-containing protein 1 (CDCP1) using ADCs and assessed the efficacy of CDCP1-directed ADCs against a range of malignant tumors. Methods: CDCP1 mRNA expression was evaluated using large transcriptomic datasets of normal/tumor samples for 23 types of cancer and 15 other normal organs, and CDCP1 protein expression was examined in 34 normal tissues, >300 samples from six types of cancer, and in 49 cancer cell lines. A recombinant human/mouse chimeric anti-CDCP1 antibody (ch10D7) was labelled with 89Zirconium or monomethyl auristatin E (MMAE) and tested in multiple pre-clinical cancer models including 36 cancer cell lines and three mouse xenograft models. Results: Analysis of CDCP1 expression indicates elevated CDCP1 expression in the majority of the cancers and restricted expression in normal human tissues. Antibody ch10D7 demonstrates a high affinity and specificity for CDCP1 inducing cell signalling via Src accompanied by rapid internalization of ch10D7/CDCP1 complexes in cancer cells. 89Zirconium-labelled ch10D7 accumulates in CDCP1 expressing cells enabling detection of pancreatic cancer xenografts in mice by PET imaging. Cytotoxicity of MMAE-labelled ch10D7 against kidney, colorectal, lung, ovarian, pancreatic and prostate cancer cells in vitro, correlates with the level of CDCP1 on the plasma membrane. ch10D7-MMAE displays robust anti-tumor effects against mouse xenograft models of pancreatic, colorectal and ovarian cancer. Conclusion: CDCP1 directed imaging agents will be useful for selecting cancer patients for personalized treatment with cytotoxin-loaded CDCP1 targeting agents including antibody-drug conjugates.
Assuntos
Antineoplásicos , Neoplasias Colorretais , Imunoconjugados , Masculino , Feminino , Humanos , Animais , Camundongos , Imunoconjugados/farmacologia , Zircônio , Linhagem Celular Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Citotoxinas , RNA Mensageiro , Antígenos de Neoplasias , Moléculas de Adesão CelularRESUMO
CUB-domain containing protein 1 (CDCP1) is a type I transmembrane glycoprotein that is upregulated in malignancies of the breast, lung, colorectum, ovary, kidney, liver, pancreas, and hematopoietic system. Here, we discuss CDCP1 as an important hub for oncogenic signaling and its key roles in malignant transformation and summarize approaches focused on exploiting it for cancer diagnosis and therapy. Elevated levels of CDCP1 are associated with progressive disease and markedly poorer survival. Predominantly located on the cell surface, CDCP1 lies at the nexus of key tumorigenic and metastatic signaling cascades, including the SRC/PKCδ, PI3K/AKT, WNT, and RAS/ERK axes, the oxidative pentose phosphate pathway, and fatty acid oxidation, making important functional contributions to cancer cell survival and growth, metastasis, and treatment resistance. These findings have stimulated the development of agents that target CDCP1 for detection and treatment of a range of cancers, and results from preclinical models suggest that these approaches could be efficacious and have manageable toxicity profiles.
Assuntos
Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Neoplasias/diagnóstico , Neoplasias/metabolismo , Transdução de Sinais , Animais , Antígenos de Neoplasias/química , Biomarcadores Tumorais/química , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Moléculas de Adesão Celular/antagonistas & inibidores , Moléculas de Adesão Celular/química , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Humanos , Terapia de Alvo Molecular/métodos , Neoplasias/tratamento farmacológico , Prognóstico , Domínios Proteicos , Transdução de Sinais/efeitos dos fármacosRESUMO
Colorectal cancer (CRC) is the third most common malignancy in the world, with 22% of patients presenting with metastatic disease and a further 50% destined to develop metastasis. Molecular imaging uses antigen-specific ligands conjugated to radionuclides to detect and characterise primary cancer and metastases. Expression of the cell surface protein CDCP1 is increased in CRC, and here we sought to assess whether it is a suitable molecular imaging target for the detection of this cancer. CDCP1 expression was assessed in CRC cell lines and a patient-derived xenograft to identify models suitable for evaluation of radio-labelled 10D7, a CDCP1-targeted, high-affinity monoclonal antibody, for preclinical molecular imaging. Positron emission tomography-computed tomography was used to compare zirconium-89 (89Zr)-10D7 avidity to a nonspecific, isotype control 89Zr-labelled IgGκ1 antibody. The specificity of CDCP1-avidity was further confirmed using CDCP1 silencing and blocking models. Our data indicate high avidity and specificity for of 89Zr-10D7 in CDCP1 expressing tumors at. Significantly higher levels than normal organs and blood, with greatest tumor avidity observed at late imaging time points. Furthermore, relatively high avidity is detected in high CDCP1 expressing tumors, with reduced avidity where CDCP1 expression was knocked down or blocked. The study supports CDCP1 as a molecular imaging target for CRC in preclinical PET-CT models using the radioligand 89Zr-10D7.
Assuntos
Antígenos de Neoplasias/genética , Moléculas de Adesão Celular/genética , Neoplasias Colorretais/genética , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Radioisótopos/farmacologia , Zircônio/farmacologia , Animais , Antígenos de Neoplasias/isolamento & purificação , Moléculas de Adesão Celular/isolamento & purificação , Neoplasias Colorretais/diagnóstico por imagem , Neoplasias Colorretais/patologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HCT116 , Xenoenxertos , Humanos , Ligantes , CamundongosRESUMO
Tyrosine kinase inhibitors (TKIs) are the first-line therapy for non-small-cell lung cancers (NSCLC) that harbour sensitising mutations within the epidermal growth factor receptor (EGFR). However, resistance remains a key issue, with tumour relapse likely to occur. We have previously identified that cell division cycle-associated protein 3 (CDCA3) is elevated in adenocarcinoma (LUAD) and correlates with sensitivity to platinum-based chemotherapy. Herein, we explored whether CDCA3 levels were associated with EGFR mutant LUAD and TKI response. We demonstrate that in a small-cohort tissue microarray and in vitro LUAD cell line panel, CDCA3 protein levels are elevated in EGFR mutant NSCLC as a result of increased protein stability downstream of receptor tyrosine kinase signalling. Here, CDCA3 protein levels correlated with TKI potency, whereby CDCA3high EGFR mutant NSCLC cells were most sensitive. Consistently, ectopic overexpression or inhibition of casein kinase 2 using CX-4945, which pharmacologically prevents CDCA3 degradation, upregulated CDCA3 levels and the response of T790M(+) H1975 cells and two models of acquired resistance to TKIs. Accordingly, it is possible that strategies to upregulate CDCA3 levels, particularly in CDCA3low tumours or upon the emergence of therapy resistance, might improve the response to EGFR TKIs and benefit patients.
RESUMO
Once thought to be exclusively a storage hub for glucose, glycogen is now known to be essential in a range of physiological processes and pathological conditions. Glycogen lies at the nexus of diverse processes that promote malignancy, including proliferation, migration, invasion, and chemoresistance of cancer cells. It is also implicated in processes associated with the tumor microenvironment such as immune cell effector function and crosstalk with cancer-associated fibroblasts to promote metastasis. The enzymes of glycogen metabolism are dysregulated in a wide variety of malignancies, including cancers of the kidney, ovary, lung, bladder, liver, blood, and breast. Understanding and targeting glycogen metabolism in cancer presents a promising but under-explored therapeutic avenue. In this review, we summarize the current literature on the role of glycogen in cancer progression and discuss its potential as a therapeutic target for cancer treatment.
RESUMO
CUB-domain containing protein 1 (CDCP1) is a cancer associated cell surface protein that amplifies pro-tumorigenic signalling by other receptors including EGFR and HER2. Its potential as a cancer target is supported by studies showing that anti-CDCP1 antibodies inhibit cell migration and survival in vitro, and tumor growth and metastasis in vivo. Here we characterize two anti-CDCP1 antibodies, focusing on immuno-conjugates of one of these as a tool to detect and inhibit ovarian cancer. Methods: A panel of ovarian cancer cell lines was examined for cell surface expression of CDCP1 and loss of expression induced by anti-CDCP1 antibodies 10D7 and 41-2 using flow cytometry and Western blot analysis. Surface plasmon resonance analysis and examination of truncation mutants was used to analyse the binding properties of the antibodies for CDCP1. Live-cell spinning-disk confocal microscopy of GFP-tagged CDCP1 was used to track internalization and intracellular trafficking of CDCP1/antibody complexes. In vivo, zirconium 89-labelled 10D7 was detected by positron-emission tomography imaging, of an ovarian cancer patient-derived xenograft grown intraperitoneally in mice. The efficacy of cytotoxin-conjugated 10D7 was examined against ovarian cancer cells in vitro and in vivo. Results: Our data indicate that each antibody binds with high affinity to the extracellular domain of CDCP1 causing rapid internalization of the receptor/antibody complex and degradation of CDCP1 via processes mediated by the kinase Src. Highlighting the potential clinical utility of CDCP1, positron-emission tomography imaging, using zirconium 89-labelled 10D7, was able to detect subcutaneous and intraperitoneal xenograft ovarian cancers in mice, including small (diameter <3 mm) tumor deposits of an ovarian cancer patient-derived xenograft grown intraperitoneally in mice. Furthermore, cytotoxin-conjugated 10D7 was effective at inhibiting growth of CDCP1-expressing ovarian cancer cells in vitro and in vivo. Conclusions: These data demonstrate that CDCP1 internalizing antibodies have potential for killing and detection of CDCP1 expressing ovarian cancer cells.
Assuntos
Moléculas de Adesão Celular/antagonistas & inibidores , Imunoconjugados/imunologia , Proteínas de Membrana/metabolismo , Neoplasias Ovarianas/metabolismo , Ressonância de Plasmônio de Superfície/métodos , Animais , Antígenos de Neoplasias/imunologia , Moléculas de Adesão Celular/imunologia , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Movimento Celular/imunologia , Feminino , Camundongos , Modelos Animais , Tomografia por Emissão de Pósitrons/métodos , Radioisótopos/química , Radioisótopos/metabolismo , Transplante Heterólogo/métodos , Zircônio/química , Zircônio/metabolismo , Quinases da Família src/metabolismoRESUMO
Background: CUB domain-containing protein 1 (CDCP1) is a cell surface receptor regulating key signalling pathways in malignant cells. CDCP1 has been proposed as a molecular target to abrogate oncogenic signalling pathways and specifically deliver anti-cancer agents to tumors. However, the development of CDCP1-targeting agents has been questioned by its frequent proteolytic processing which was thought to result in shedding of the CDCP1 extracellular domain limiting its targetability. In this study, we investigated the relevance of targeting CDCP1 in the context of pancreatic ductal adenocarcinoma (PDAC) and assess the impact of CDCP1 proteolysis on the effectiveness of CDCP1 targeting agents. Methods: The involvement of CDCP1 in PDAC progression was assessed by association analysis in several PDAC cohorts and the proteolytic processing of CDCP1 was evaluated in PDAC cell lines and patient-derived cells. The consequences of CDCP1 proteolysis on its targetability in PDAC cells was assessed using immunoprecipitation, immunostaining and biochemical assays. The involvement of CDCP1 in PDAC progression was examined by loss-of-function in vitro and in vivo experiments employing PDAC cells expressing intact or cleaved CDCP1. Finally, we generated antibody-based imaging and therapeutic agents targeting CDCP1 to demonstrate the feasibility of targeting this receptor for detection and treatment of PDAC tumors. Results: High CDCP1 expression in PDAC is significantly associated with poorer patient survival. In PDAC cells proteolysis of CDCP1 does not always result in the shedding of CDCP1-extracellular domain which can interact with membrane-bound CDCP1 allowing signal transduction between the different CDCP1-fragments. Targeting CDCP1 impairs PDAC cell functions and PDAC tumor growth independently of CDCP1 cleavage status. A CDCP1-targeting antibody is highly effective at delivering imaging radionuclides and cytotoxins to PDAC cells allowing specific detection of tumors by PET/CT imaging and superior anti-tumor effects compared to gemcitabine in in vivo models. Conclusion: Independent of its cleavage status, CDCP1 exerts oncogenic functions in PDAC and has significant potential to be targeted for improved radiological staging and treatment of this cancer. Its elevated expression by most PDAC tumors and lack of expression by normal pancreas and other major organs, suggest that targeting CDCP1 could benefit a significant proportion of PDAC patients. These data support the further development of CDCP1-targeting agents as personalizable tools for effective imaging and treatment of PDAC.
Assuntos
Antígenos de Neoplasias/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Moléculas de Adesão Celular/metabolismo , Neoplasias Pancreáticas/metabolismo , Animais , Biomarcadores Tumorais/metabolismo , Carcinoma Ductal Pancreático/terapia , Linhagem Celular Tumoral , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Neoplasias Pancreáticas/terapia , Medicina de Precisão , ProteóliseRESUMO
High stage and recurrent ovarian clear cell carcinoma (OCC) are associated with poor prognosis and resistance to chemotherapy. A distinguishing histological feature of OCC is abundant cytoplasmic stores of glucose, in the form of glycogen, that can be mobilized for cellular metabolism. Here, we report the effect on preclinical models of OCC of disrupting glycogen utilization using the glucose analogue 2-deoxy-D-glucose (2DG). At concentrations significantly lower than previously reported for other cancers, 2DG markedly improves the efficacy in vitro of carboplatin chemotherapy against chemo-sensitive TOV21G and chemo-resistant OVTOKO OCC cell lines, and this is accompanied by the depletion of glycogen. Of note, 2DG doses-of more than 10-fold lower than previously reported for other cancers-significantly improve the efficacy of carboplatin against cell line and patient-derived xenograft models in mice that mimic the chemo-responsiveness of OCC. These findings are encouraging, in that 2DG doses, which are substantially lower than previously reported to cause adverse events in cancer patients, can safely and significantly improve the efficacy of carboplatin against OCC. Our results thus justify clinical trials to evaluate whether low dose 2DG improves the efficacy of carboplatin in OCC patients.