RESUMO
Although RAF monomer inhibitors (type I.5, BRAF(V600)) are clinically approved for the treatment of BRAFV600-mutant melanoma, they are ineffective in non-BRAFV600 mutant cells1-3. Belvarafenib is a potent and selective RAF dimer (type II) inhibitor that exhibits clinical activity in patients with BRAFV600E- and NRAS-mutant melanomas. Here we report the first-in-human phase I study investigating the maximum tolerated dose, and assessing the safety and preliminary efficacy of belvarafenib in BRAFV600E- and RAS-mutated advanced solid tumours (NCT02405065, NCT03118817). By generating belvarafenib-resistant NRAS-mutant melanoma cells and analysing circulating tumour DNA from patients treated with belvarafenib, we identified new recurrent mutations in ARAF within the kinase domain. ARAF mutants conferred resistance to belvarafenib in both a dimer- and a kinase activity-dependent manner. Belvarafenib induced ARAF mutant dimers, and dimers containing mutant ARAF were active in the presence of inhibitor. ARAF mutations may serve as a general resistance mechanism for RAF dimer inhibitors as the mutants exhibit reduced sensitivity to a panel of type II RAF inhibitors. The combination of RAF plus MEK inhibition may be used to delay ARAF-driven resistance and suggests a rational combination for clinical use. Together, our findings reveal specific and compensatory functions for the ARAF isoform and implicate ARAF mutations as a driver of resistance to RAF dimer inhibitors.
Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Melanoma/tratamento farmacológico , Melanoma/genética , Mutação , Proteínas Proto-Oncogênicas A-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas A-raf/genética , Quinases raf/antagonistas & inibidores , Animais , Linhagem Celular , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Humanos , Melanoma/patologia , Camundongos , Multimerização Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas A-raf/química , Quinases raf/químicaRESUMO
Multiple myeloma (MM) arises from malignant immunoglobulin (Ig)-secreting plasma cells and remains an incurable, often lethal disease despite therapeutic advances. The unfolded-protein response sensor IRE1α supports protein secretion by deploying a kinase-endoribonuclease module to activate the transcription factor XBP1s. MM cells may co-opt the IRE1α-XBP1s pathway; however, the validity of IRE1α as a potential MM therapeutic target is controversial. Genetic disruption of IRE1α or XBP1s, or pharmacologic IRE1α kinase inhibition, attenuated subcutaneous or orthometastatic growth of MM tumors in mice and augmented efficacy of two established frontline antimyeloma agents, bortezomib and lenalidomide. Mechanistically, IRE1α perturbation inhibited expression of key components of the endoplasmic reticulum-associated degradation machinery, as well as secretion of Ig light chains and of cytokines and chemokines known to promote MM growth. Selective IRE1α kinase inhibition reduced viability of CD138+ plasma cells while sparing CD138- cells derived from bone marrows of newly diagnosed or posttreatment-relapsed MM patients, in both US- and European Union-based cohorts. Effective IRE1α inhibition preserved glucose-induced insulin secretion by pancreatic microislets and viability of primary hepatocytes in vitro, as well as normal tissue homeostasis in mice. These results establish a strong rationale for developing kinase-directed inhibitors of IRE1α for MM therapy.
Assuntos
Endorribonucleases/genética , Mieloma Múltiplo/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/genética , Idoso , Animais , Bortezomib/farmacologia , Estresse do Retículo Endoplasmático/genética , Endorribonucleases/antagonistas & inibidores , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Lenalidomida/farmacologia , Masculino , Camundongos , Pessoa de Meia-Idade , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Resposta a Proteínas não Dobradas/genética , Proteína 1 de Ligação a X-Box/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Whereas cancers grow within host tissues and evade host immunity through immune-editing and immunosuppression, tumours are rarely transmissible between individuals. Much like transplanted allogeneic organs, allogeneic tumours are reliably rejected by host T cells, even when the tumour and host share the same major histocompatibility complex alleles, the most potent determinants of transplant rejection. How such tumour-eradicating immunity is initiated remains unknown, although elucidating this process could provide the basis for inducing similar responses against naturally arising tumours. Here we find that allogeneic tumour rejection is initiated in mice by naturally occurring tumour-binding IgG antibodies, which enable dendritic cells (DCs) to internalize tumour antigens and subsequently activate tumour-reactive T cells. We exploited this mechanism to treat autologous and autochthonous tumours successfully. Either systemic administration of DCs loaded with allogeneic-IgG-coated tumour cells or intratumoral injection of allogeneic IgG in combination with DC stimuli induced potent T-cell-mediated antitumour immune responses, resulting in tumour eradication in mouse models of melanoma, pancreas, lung and breast cancer. Moreover, this strategy led to eradication of distant tumours and metastases, as well as the injected primary tumours. To assess the clinical relevance of these findings, we studied antibodies and cells from patients with lung cancer. T cells from these patients responded vigorously to autologous tumour antigens after culture with allogeneic-IgG-loaded DCs, recapitulating our findings in mice. These results reveal that tumour-binding allogeneic IgG can induce powerful antitumour immunity that can be exploited for cancer immunotherapy.
Assuntos
Anticorpos Antineoplásicos/imunologia , Antígenos de Neoplasias/imunologia , Células Dendríticas/imunologia , Imunoglobulina G/imunologia , Neoplasias/imunologia , Linfócitos T/imunologia , Animais , Anticorpos Antineoplásicos/administração & dosagem , Antígenos CD40/metabolismo , Modelos Animais de Doenças , Feminino , Imunoglobulina G/administração & dosagem , Isoanticorpos/administração & dosagem , Isoanticorpos/imunologia , Ativação Linfocitária/imunologia , Masculino , Camundongos , Metástase Neoplásica , Transplante de Neoplasias/imunologia , Neoplasias/patologia , Receptores de IgG/imunologia , Fator de Necrose Tumoral alfa/imunologiaRESUMO
The cysteine cathepsins are a group of 11 proteases whose function was originally believed to be the degradation of endocytosed material with a high degree of redundancy. However, it has become clear that these enzymes are also important regulators of both health and disease. Thus, selective tools that can discriminate between members of this highly related class of enzymes will be critical to further delineate the unique biological functions of individual cathepsins. Here we present the design and synthesis of a near-infrared quenched activity-based probe (qABP) that selectively targets cathepsin S which is highly expressed in immune cells. Importantly, this high degree of selectivity is retained both in vitro and in vivo. In combination with a new green-fluorescent pan-reactive cysteine cathepsin qABP we performed dual color labeling studies in bone marrow-derived immune cells and identified vesicles containing exclusively cathepsin S activity. This observation demonstrates the value of our complementary cathepsin probes and provides evidence for the existence of specific localization of cathepsin S activity in dendritic cells.
Assuntos
Catepsinas/química , Catepsinas/metabolismo , Desenho de Fármacos , Corantes Fluorescentes/química , Raios Infravermelhos , Imagem Óptica/métodos , Animais , Cor , Células Dendríticas/enzimologia , Humanos , Neoplasias Mamárias Experimentais/enzimologia , Camundongos , Células RAW 264.7 , Especificidade por SubstratoRESUMO
The lack of selective and safe in vivo IRE1α tool molecules has limited the evaluation of IRE1α as a viable target to treat multiple myeloma. Focus on improving the physicochemical properties of a literature compound by decreasing lipophilicity, molecular weight, and basicity allowed the discovery of a novel series with a favorable in vitro safety profile and good oral exposure. These efforts culminated in the identification of a potent and selective in vivo tool compound, G-5758, that was well tolerated following multiday oral administration of doses up to 500 mg/kg. G-5758 demonstrated comparable pharmacodynamic effects to induced IRE1 knockdown as measured by XBP1s levels in a multiple myeloma model (KMS-11).
Assuntos
Endorribonucleases , Mieloma Múltiplo , Proteínas Serina-Treonina Quinases , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/patologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Humanos , Administração Oral , Endorribonucleases/antagonistas & inibidores , Endorribonucleases/metabolismo , Animais , Descoberta de Drogas , Camundongos , Linhagem Celular Tumoral , Relação Estrutura-Atividade , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Ratos , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/uso terapêutico , Inibidores de Proteínas Quinases/farmacocinética , Técnicas de Silenciamento de Genes , Proteína 1 de Ligação a X-Box/metabolismo , Proteína 1 de Ligação a X-Box/genéticaRESUMO
The cysteine cathepsins are a family of proteases that play important roles in both normal cellular physiology and many human diseases. In cancer, the activity of many of the cysteine cathepsins is upregulated and can be exploited for tumor imaging. Here we present the design and synthesis of a new class of quenched fluorescent activity-based probes (qABPs) containing a phenoxymethyl ketone (PMK) electrophile. These reagents show enhanced in vivo properties and broad reactivity resulting in dramatically improved labeling and tumor imaging properties compared to those of previously reported ABPs.
Assuntos
Neoplasias da Mama/diagnóstico , Neoplasias da Mama/enzimologia , Mama/patologia , Cisteína Proteases/análise , Corantes Fluorescentes/química , Animais , Mama/enzimologia , Neoplasias da Mama/patologia , Linhagem Celular , Células Cultivadas , Cisteína/metabolismo , Cisteína Proteases/metabolismo , Feminino , Corantes Fluorescentes/síntese química , Humanos , Cetonas/síntese química , Cetonas/química , Camundongos , Imagem Óptica/métodosRESUMO
Tumor-initiating cells (TICs) are a subtype of tumor cells believed to be critical for initiating tumorigenesis. We sought to determine the angiogenic properties of TICs in different tumor types including U-87MG (glioblastoma), HT29 (colon), MCF7 (breast), A549 (non-small-cell lung), and PANC1 (pancreatic) cancers. Long-term cultures grown either as monolayers ("TIC-low") or as nonadherent tumor spheres ("TIC-high") were generated. The TIC-high fractions exhibited increased expression of stem cell surface markers, high aldehyde dehydrogenase activity, high expression of p21, and resistance to standard chemotherapy in comparison to TIC-low fractions. Furthermore, TICs from U-87MG and HT29 but not from MCF7, A549, and PANC1 tumor types possess increased angiogenic activity. Consequently, the efficacy of vascular endothelial growth factor-A (VEGF-A) neutralizing antibody is limited only to those tumors that are dependent on VEGF-A activity. In addition, such therapy had little or reversed antiangiogenic effects on tumors that do not necessarily rely on VEGF-dependent angiogenesis. Differential angiogenic activity and antiangiogenic therapy sensitivity were also observed in TICs of the same tumor type, suggesting redundant angiogenic pathways. Collectively, our results suggest that the efficacy of antiangiogenic drugs is dependent on the angiogenic properties of TICs and, therefore, can serve as a possible biomarker to predict antiangiogenic treatment efficacy.
Assuntos
Inibidores da Angiogênese/farmacologia , Neoplasias/irrigação sanguínea , Neoplasias/tratamento farmacológico , Células-Tronco Neoplásicas/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Células HT29 , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Immunoblotting , Células MCF-7 , Camundongos , Camundongos Nus , Neoplasias/patologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/patologia , Transfecção , Transplante HeterólogoRESUMO
Cyclin-dependent kinases (CDKs) are key regulators of the cell cycle and are frequently altered in cancer cells, thereby leading to uncontrolled proliferation. In this context, CDK2 has emerged as an appealing target for anticancer drug development. Herein, we describe the discovery of a series of selective small molecule inhibitors of CDK2 beginning with historical compounds from our ERK2 program (e.g., compound 6). Structure-based drug design led to the potent and selective tool compound 32, where excellent selectivity against ERK2 and CDK4 was achieved by filling the lipophilic DFG-1 pocket and targeting interactions with CDK2-specific lower hinge binding residues, respectively. Compound 32 demonstrated 112% tumor growth inhibition in mice bearing OVCAR3 tumors with 50 mg/kg bis in die (BID) oral dosing.
RESUMO
KRAS, which is mutated in â¼30% of all cancers, activates the RAF-MEK-ERK signaling cascade. CRAF is required for growth of KRAS mutant lung tumors, but the requirement for CRAF kinase activity is unknown. Here, we show that subsets of KRAS mutant tumors are dependent on CRAF for growth. Kinase-dead but not dimer-defective CRAF rescues growth inhibition, suggesting that dimerization but not kinase activity is required. Quantitative proteomics demonstrates increased levels of CRAF:ARAF dimers in KRAS mutant cells, and depletion of both CRAF and ARAF rescues the CRAF-loss phenotype. Mechanistically, CRAF depletion causes sustained ERK activation and induction of cell-cycle arrest, while treatment with low-dose MEK or ERK inhibitor rescues the CRAF-loss phenotype. Our studies highlight the role of CRAF in regulating MAPK signal intensity to promote tumorigenesis downstream of mutant KRAS and suggest that disrupting CRAF dimerization or degrading CRAF may have therapeutic benefit.
Assuntos
Carcinogênese/metabolismo , Dimerização , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Animais , Carcinogênese/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Fosforilação/fisiologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Proteínas ras/genéticaRESUMO
The development of highly sensitive fluorescent probes in combination with innovative optical techniques is a promising strategy for intravital noninvasive quantitative imaging. Cyanine fluorochromes belong to a superfamily of dyes that have attracted substantial attention in probe design for molecular imaging. We have developed a novel paradigm to introduce a Turn-ON mechanism in cyanine molecules, based on a distinctive change in their π-electrons system. Our new cyanine fluorochrome is synthesized through a simple two-step procedure and has an unprecedented high fluorescence quantum yield of 16% and large extinction coefficient of 52,000 M(-1)cm(-1). The synthetic strategy allows one to prepare probes for various analytes by introducing a specific triggering group on the probe molecule. The probe was equipped with a corresponding trigger and demonstrated efficient imaging of endogenous hydrogen peroxide, produced in an acute lipopolysaccharide-induced inflammation model in mice. This approach provides, for the first time, an available methodology to prepare modular molecular Turn-ON probes that can release an active cyanine fluorophore upon reaction with specific analyte.
Assuntos
Carbocianinas/metabolismo , Corantes Fluorescentes/metabolismo , Peróxido de Hidrogênio/metabolismo , Raios Infravermelhos , Imagem Molecular/métodos , Fenômenos Ópticos , Animais , CamundongosRESUMO
Polymer therapeutics have shown promise as tumor-targeted drug delivery systems in mice. The multivalency of polymers allows the attachment of different functional agents to a polymeric backbone, including chemotherapeutic and antiangiogenic drugs, as well as targeting moieties, such as the bone-targeting agent alendronate (ALN). We previously reported the conjugation of ALN and the chemotherapeutic drug paclitaxel (PTX) with N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer. The in vitro physicochemical properties, cancer cytotoxicity and antiangiogenic activity of HPMA copolymer-PTX-ALN conjugate were extensively characterized. The reported results warranted in vivo evaluations of the conjugate. In this manuscript, we evaluated the in vivo anticancer and antiangiogenic activity of HPMA copolymer-PTX-ALN conjugate. The conjugate exhibited an antiangiogenic effect by decreasing microvessel density (MVD), and inducing apoptotic circulating endothelial cells (CEC) following treatment of the mice. Using intravital imaging system and mCherry-labeled breast cancer cell lines, we were able to monitor noninvasively the progression of orthotopic metastatic tumors injected into the tibia of the mice. HPMA copolymer-PTX-ALN conjugate showed the greatest antitumor efficacy on mCherry-labeled 4T1 mammary adenocarcinoma inoculated into the tibia, as compared with PTX alone or in combination with ALN. Treatment with the bone-targeted polymeric conjugate demonstrated improved efficacy, was better tolerated, and was more easily administered intravenously than the clinically used PTX formulated in Cremophor/ethanol.
Assuntos
Acrilamidas/química , Alendronato/química , Alendronato/uso terapêutico , Inibidores da Angiogênese/uso terapêutico , Antineoplásicos/uso terapêutico , Neoplasias Ósseas/tratamento farmacológico , Neoplasias da Mama/tratamento farmacológico , Paclitaxel/química , Paclitaxel/uso terapêutico , Inibidores da Angiogênese/química , Inibidores da Angiogênese/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Neoplasias Ósseas/secundário , Neoplasias da Mama/complicações , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Feminino , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Contagem de Leucócitos , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Cancer cells exploit the unfolded protein response (UPR) to mitigate endoplasmic reticulum (ER) stress caused by cellular oncogene activation and a hostile tumor microenvironment (TME). The key UPR sensor IRE1α resides in the ER and deploys a cytoplasmic kinase-endoribonuclease module to activate the transcription factor XBP1s, which facilitates ER-mediated protein folding. Studies of triple-negative breast cancer (TNBC)-a highly aggressive malignancy with a dismal posttreatment prognosis-implicate XBP1s in promoting tumor vascularization and progression. However, it remains unknown whether IRE1α adapts the ER in TNBC cells and modulates their TME, and whether IRE1α inhibition can enhance antiangiogenic therapy-previously found to be ineffective in patients with TNBC. To gauge IRE1α function, we defined an XBP1s-dependent gene signature, which revealed significant IRE1α pathway activation in multiple solid cancers, including TNBC. IRE1α knockout in TNBC cells markedly reversed substantial ultrastructural expansion of their ER upon growth in vivo. IRE1α disruption also led to significant remodeling of the cellular TME, increasing pericyte numbers while decreasing cancer-associated fibroblasts and myeloid-derived suppressor cells. Pharmacologic IRE1α kinase inhibition strongly attenuated growth of cell line-based and patient-derived TNBC xenografts in mice and synergized with anti-VEGFA treatment to cause tumor stasis or regression. Thus, TNBC cells critically rely on IRE1α to adapt their ER to in vivo stress and to adjust the TME to facilitate malignant growth. TNBC reliance on IRE1α is an important vulnerability that can be uniquely exploited in combination with antiangiogenic therapy as a promising new biologic approach to combat this lethal disease. SIGNIFICANCE: Pharmacologic IRE1α kinase inhibition reverses ultrastructural distension of the ER, normalizes the tumor vasculature, and remodels the cellular TME, attenuating TNBC growth in mice.
Assuntos
Inibidores da Angiogênese/farmacologia , Antineoplásicos Imunológicos/farmacologia , Estresse do Retículo Endoplasmático/fisiologia , Endorribonucleases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Neoplasias de Mama Triplo Negativas/terapia , Animais , Antineoplásicos Imunológicos/imunologia , Linhagem Celular Tumoral , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Endorribonucleases/genética , Feminino , Técnicas de Inativação de Genes , Humanos , Camundongos , Camundongos SCID , Neovascularização Patológica/terapia , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/genética , Neoplasias de Mama Triplo Negativas/irrigação sanguínea , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Microambiente Tumoral , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/imunologia , Proteína 1 de Ligação a X-Box/antagonistas & inibidores , Proteína 1 de Ligação a X-Box/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The development of targeting approaches to selectively release chemotherapeutic drugs into malignant tissue is a major challenge in anticancer therapy. We have synthesized an N-(2-hydroxypropyl)-methacrylamide (HPMA) copolymer-drug conjugate with an AB(3) self-immolative dendritic linker. HPMA copolymers are known to accumulate selectively in tumors. The water-soluble polymer-drug conjugate was designed to release a triple payload of the hydrophobic drug paclitaxel as a result of cleavage by the endogenous enzyme cathepsin B. The polymer-drug conjugate exhibited enhanced cytotoxicity on murine prostate adenocarcinoma (TRAMP C2) cells in comparison to a classic monomeric drug-polymer conjugate.
Assuntos
Acrilamidas/administração & dosagem , Antineoplásicos/administração & dosagem , Proliferação de Células/efeitos dos fármacos , Dendrímeros/administração & dosagem , Pró-Fármacos/administração & dosagem , Acrilamidas/química , Adenocarcinoma/tratamento farmacológico , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Dendrímeros/química , Sistemas de Liberação de Medicamentos , Masculino , Camundongos , Paclitaxel , Pró-Fármacos/química , Pró-Fármacos/uso terapêutico , Neoplasias da Próstata/tratamento farmacológicoRESUMO
A polymer therapeutic designed for combination anticancer and antiangiogenic therapy inhibited the proliferation of prostate carcinoma cells and the proliferation, migration, and tube-formation of endothelial cells. The nanoconjugate was formed from an N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer, the bisphosphonate alendronate (for bone targeting), and the chemotherapy agent paclitaxel (PTX), which is cleaved by cathepsin B (see scheme).
Assuntos
Acrilamidas/química , Inibidores da Angiogênese/química , Antineoplásicos/química , Paclitaxel/análogos & derivados , Acrilamidas/síntese química , Alendronato/química , Inibidores da Angiogênese/síntese química , Inibidores da Angiogênese/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/secundário , Hidrocarbonetos Aromáticos com Pontes/química , Catepsina B/metabolismo , Células Cultivadas , Humanos , Paclitaxel/síntese química , Paclitaxel/química , Polímeros/química , Taxoides/química , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Resistance invariably develops to antiandrogen therapies used to treat newly diagnosed prostate cancers, but effective treatments for castration-resistant disease remain elusive. Here, we report that the transcriptional coactivator CBP/p300 is required to maintain the growth of castration-resistant prostate cancer. To exploit this vulnerability, we developed a novel small-molecule inhibitor of the CBP/p300 bromodomain that blocks prostate cancer growth in vitro and in vivo Molecular dissection of the consequences of drug treatment revealed a critical role for CBP/p300 in histone acetylation required for the transcriptional activity of the androgen receptor and its target gene expression. Our findings offer a preclinical proof of concept for small-molecule therapies to target the CBP/p300 bromodomain as a strategy to treat castration-resistant prostate cancer. Cancer Res; 77(20); 5564-75. ©2017 AACR.
Assuntos
Proteína p300 Associada a E1A/antagonistas & inibidores , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proteína p300 Associada a E1A/deficiência , Proteína p300 Associada a E1A/genética , Proteína p300 Associada a E1A/metabolismo , Feminino , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Masculino , Camundongos , Camundongos SCID , Terapia de Alvo Molecular , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/metabolismo , Neoplasias de Próstata Resistentes à Castração/patologia , Domínios Proteicos , Distribuição Aleatória , Receptores Androgênicos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transfecção , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Active enzymes, such as proteases, often serve as valuable biomarkers for various disease pathologies. Therefore, methods to detect specific enzyme activities in biological samples can provide information to guide disease detection and diagnosis and to increase our understanding of the biological roles of specific enzyme targets. In this protocol, we outline methods for the topical application of fluorescently quenched activity-based probes (qABPs) to fresh-frozen tissue samples. This technique enables rapid imaging of enzyme activity at cellular resolution, and it can be combined with antibody labeling for immunodiagnosis. In this method, fresh-frozen tissue sections are fixed, incubated with the probe and imaged using fluorescence microscopy. This provides an advance over classical immunohistochemistry (IHC) in that it is rapid (4-8 h) and inexpensive, and it provides information on enzyme activity. Furthermore, it can be used with any of the growing number of fluorescent ABPs to provide data for more effective disease monitoring and diagnosis.
Assuntos
Criopreservação , Corantes Fluorescentes/metabolismo , Peptídeo Hidrolases/metabolismo , Coloração e Rotulagem/métodos , Ensaios Enzimáticos , Humanos , Imuno-Histoquímica , Microscopia de Fluorescência , Coloração e Rotulagem/economia , Temperatura , Fixação de Tecidos , Carga TumoralRESUMO
Colon tumors arise in a stepwise fashion from either discrete genetic perturbations or epigenetic dysregulation. To uncover the key epigenetic regulators that drive colon cancer growth, we used a CRISPR loss-of-function screen and identified a number of essential genes, including the bromodomain and extraterminal (BET) protein BRD4. We found that BRD4 is critical for colon cancer proliferation, and its knockdown led to differentiation effects in vivo. JQ1, a BET inhibitor, preferentially reduced growth in a subset of epigenetically dysregulated colon cancers characterized by the CpG island methylator phenotype (CIMP). Integrated transcriptomic and genomic analyses defined a distinct superenhancer in CIMP+ colon cancers that regulates cMYC transcription. We found that the long noncoding RNA colon cancer-associated transcript 1 (CCAT1) is transcribed from this superenhancer and is exquisitely sensitive to BET inhibition. Concordantly, cMYC transcription and cell growth were tightly correlated with the presence of CCAT1 RNA in a variety of tumor types. Taken together, we propose that CCAT1 is a clinically tractable biomarker for identifying patients who are likely to benefit from BET inhibitors.
Assuntos
Biomarcadores Tumorais/metabolismo , Proliferação de Células , Proteínas Nucleares/metabolismo , RNA Longo não Codificante/metabolismo , RNA Neoplásico/metabolismo , Fatores de Transcrição/metabolismo , Animais , Azepinas/farmacologia , Biomarcadores Tumorais/genética , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Neoplasias Colorretais , Ilhas de CpG , Metilação de DNA/efeitos dos fármacos , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Camundongos Nus , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Longo não Codificante/genética , RNA Neoplásico/genética , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Triazóis/farmacologiaRESUMO
PURPOSE: Early and effective detection of cancers of the gastrointestinal tract will require novel molecular probes and advances in instrumentation that can reveal functional changes in dysplastic and malignant tissues. Here, we describe adaptation of a wide-field clinical fiberscope to perform wide-field fluorescence imaging while preserving its white-light capability for the purpose of providing wide-field fluorescence imaging capability to point-of-care microscopes. PROCEDURES: We developed and used a fluorescent fiberscope to detect signals from a quenched probe, BMV109, that becomes fluorescent when cleaved by, and covalently bound to, active cathepsin proteases. Cathepsins are expressed in inflammation- and tumor-associated macrophages as well as directly from tumor cells and are a promising target for cancer imaging. The fiberscope has a 1-mm outer diameter enabling validation via endoscopic exams in mice, and therefore we evaluated topically applied BMV109 for the ability to detect colon polyps in an azoxymethane-induced colon tumor model in mice. RESULTS: This wide-field endoscopic imaging device revealed consistent and clear fluorescence signals from BMV109 that specifically localized to the polypoid regions as opposed to the normal adjacent colon tissue (p < 0.004) in the murine colon carcinoma model. CONCLUSIONS: The sensitivity of detection of BMV109 with the fluorescence fiberscope suggested utility of these tools for early detection at hard-to-reach sites. The fiberscope was designed to be used in conjunction with miniature, endoscope-compatible fluorescence microscopes for dual wide-field and microscopic cancer detection.
Assuntos
Catepsinas/metabolismo , Neoplasias do Colo/diagnóstico por imagem , Neoplasias do Colo/enzimologia , Endoscopia/instrumentação , Imagem Molecular/instrumentação , Animais , Colo/patologia , Neoplasias do Colo/patologia , Pólipos do Colo/patologia , Fluorescência , Camundongos , Microscopia ConfocalRESUMO
The presence of dormant, microscopic cancerous lesions poses a major obstacle for the treatment of metastatic and recurrent cancers. While it is well-established that microRNAs play a major role in tumorigenesis, their involvement in tumor dormancy has yet to be fully elucidated. We established and comprehensively characterized pairs of dormant and fast-growing human osteosarcoma models. Using these pairs of mouse tumor models, we identified three novel regulators of osteosarcoma dormancy: miR-34a, miR-93, and miR-200c. This report shows that loss of these microRNAs occurs during the switch from dormant avascular into fast-growing angiogenic phenotype. We validated their downregulation in patients' tumor samples compared to normal bone, making them attractive candidates for osteosarcoma therapy. Successful delivery of miRNAs is a challenge; hence, we synthesized an aminated polyglycerol dendritic nanocarrier, dPG-NH2, and designed dPG-NH2-microRNA polyplexes to target cancer. Reconstitution of these microRNAs using dPG-NH2 polyplexes into Saos-2 and MG-63 cells, which generate fast-growing osteosarcomas, reduced the levels of their target genes, MET proto-oncogene, hypoxia-inducible factor 1α, and moesin, critical to cancer angiogenesis and cancer cells' migration. We further demonstrate that these microRNAs attenuate the angiogenic capabilities of fast-growing osteosarcomas in vitro and in vivo. Treatment with each of these microRNAs using dPG-NH2 significantly prolonged the dormancy period of fast-growing osteosarcomas in vivo. Taken together, these findings suggest that nanocarrier-mediated delivery of microRNAs involved in osteosarcoma tumor-host interactions can induce a dormant-like state.
Assuntos
Antineoplásicos/química , Portadores de Fármacos/química , Glicerol/química , MicroRNAs/genética , Nanoestruturas/química , Osteossarcoma/genética , Polímeros/química , Animais , Linhagem Celular Tumoral , Descoberta de Drogas , Humanos , Camundongos , Camundongos SCID , MicroRNAs/química , Nanomedicina , Osteossarcoma/metabolismo , Proto-Oncogene MasRESUMO
Bleomycin hydrolase (BLMH) is a neutral cysteine aminopeptidase that has been ascribed roles in many physiological and pathological processes, yet its primary biological function remains enigmatic. In this work, we describe the results of screening of a library of fluorogenic substrates to identify non-natural amino acids that are optimally recognized by BLMH. This screen identified several substrates with kcat/KM values that are substantially improved over the previously reported fluorogenic substrates for this enzyme. The substrate sequences were used to design activity-based probes that showed potent labeling of recombinant BLMH as well as endogenously expressed BLMH in cell extracts, and in intact cells. Importantly, we identify potent BLMH inhibitors that are able to fully inhibit endogenous BLMH activity in intact cells. These probes and inhibitors will be valuable new reagents to study BLMH function in cellular and animal models of human diseases where BLMH is likely to be involved.