RESUMO
Homologous recombination (HR) plays an essential role in the maintenance of genome stability by promoting the repair of cytotoxic DNA double strand breaks (DSBs). More recently, the HR pathway has emerged as a core component of the response to replication stress, in part by protecting stalled replication forks from nucleolytic degradation. In that regard, the mammalian RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3) have been involved in both HR-mediated DNA repair and collapsed replication fork resolution. Still, it remains largely obscure how they participate in both processes, thereby maintaining genome stability and preventing cancer development. To gain better insight into their contribution in cellulo, we mapped the proximal interactome of the classical RAD51 paralogs using the BioID approach. Aside from identifying the well-established BCDX2 and CX3 sub-complexes, the spliceosome machinery emerged as an integral component of our proximal mapping, suggesting a crosstalk between this pathway and the RAD51 paralogs. Furthermore, we noticed that factors involved RNA metabolic pathways are significantly modulated within the BioID of the classical RAD51 paralogs upon exposure to hydroxyurea (HU), pointing towards a direct contribution of RNA processing during replication stress. Importantly, several members of these pathways have prognostic potential in breast cancer (BC), where their RNA expression correlates with poorer patient outcome. Collectively, this study uncovers novel functionally relevant partners of the different RAD51 paralogs in the maintenance of genome stability that could be used as biomarkers for the prognosis of BC.
Assuntos
Instabilidade Genômica , Rad51 Recombinase , Animais , Humanos , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Instabilidade Genômica/genética , Recombinação Homóloga/genética , Quebras de DNA de Cadeia Dupla , RNA , Reparo do DNA/genética , Mamíferos/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismoRESUMO
Tumor-associated macrophages (TAMs) continuously fine tune their immune modulatory properties, but how gene expression programs coordinate this immune cell plasticity is largely unknown. Selective mRNA translation, controlled by MNK1/MNK2 and mTOR pathways impinging on eIF4E, facilitates reshaping of proteomes without changes in abundance of corresponding mRNAs. Using polysome profiling developed for small samples we show that, during tumor growth, gene expression in TAMs is predominately modulated via mRNA-selective changes in translational efficiencies. These alterations in gene expression paralleled accumulation of antiinflammatory macrophages with augmented phosphorylation of eIF4E, a target of the MNK1 and MNK2 kinases, known to selectively modulate mRNA translation. Furthermore, suppression of the MNK2, but not the mTOR signaling pathway, reprogrammed antiinflammatory macrophages toward a proinflammatory phenotype with the ability to activate CD8+ T cells. Thus, selective changes of mRNA translation depending on MNK2 signaling represents a key node regulating macrophage antiinflammatory functions.
Assuntos
Macrófagos/imunologia , Neoplasias/imunologia , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Técnicas de Cocultura , Modelos Animais de Doenças , Fator de Iniciação 4E em Eucariotos/genética , Fator de Iniciação 4E em Eucariotos/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/imunologia , Técnicas de Silenciamento de Genes , Humanos , Células MCF-7 , Macrófagos/metabolismo , Camundongos , Camundongos Transgênicos , Naftiridinas/farmacologia , Neoplasias/genética , Neoplasias/patologia , Fosforilação/genética , Fosforilação/imunologia , Cultura Primária de Células , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Evasão Tumoral/genéticaRESUMO
Most human tumor tissues that are obtained for pathology and diagnostic purposes are formalin-fixed and paraffin-embedded (FFPE). To perform quantitative proteomics of FFPE samples, paraffin has to be removed and formalin-induced crosslinks have to be reversed prior to proteolytic digestion. A central component of almost all deparaffinization protocols is xylene, a toxic and highly flammable solvent that has been reported to negatively affect protein extraction and quantitative proteome analysis. Here, we present a 'green' xylene-free protocol for accelerated sample preparation of FFPE tissues based on paraffin-removal with hot water. Combined with tissue homogenization using disposable micropestles and a modified protein aggregation capture (PAC) digestion protocol, our workflow enables streamlined and reproducible quantitative proteomic profiling of FFPE tissue. Label-free quantitation of FFPE cores from human ductal breast carcinoma in situ (DCIS) xenografts with a volume of only 0.79 mm3 showed a high correlation between replicates (r2 = 0.992) with a median %CV of 16.9%. Importantly, this small volume is already compatible with tissue micro array (TMA) cores and core needle biopsies, while our results and its ease-of-use indicate that further downsizing is feasible. Finally, our FFPE workflow does not require costly equipment and can be established in every standard clinical laboratory.
Assuntos
Parafina , Proteômica , Biópsia com Agulha de Grande Calibre , Formaldeído , Humanos , Inclusão em Parafina , Proteoma/metabolismo , Proteômica/métodos , Fixação de TecidosRESUMO
UNLABELLED: The V proteins of paramyxoviruses control the innate immune response. In particular, the V protein of the genus Morbillivirus interferes with the signal transducer and activator of transcription 1 (STAT1), STAT2, and melanoma differentiation-associated protein 5 (mda5) signaling pathways. To characterize the contributions of these pathways to canine distemper virus (CDV) pathogenesis, we took advantage of the knowledge about the mechanisms of interaction between the measles virus V protein with these key regulators of innate immunity. We generated recombinant CDVs with V proteins unable to properly interact with STAT1, STAT2, or mda5. A virus with combined STAT2 and mda5 deficiencies was also generated, and available wild-type and V-protein-knockout viruses were used as controls. Ferrets infected with wild-type and STAT1-blind viruses developed severe leukopenia and loss of lymphocyte proliferation activity and succumbed to the disease within 14 days. In contrast, animals infected with viruses with STAT2 or mda5 defect or both STAT2 and mda5 defects developed a mild self-limiting disease similar to that associated with the V-knockout virus. This study demonstrates the importance of interference with STAT2 and mda5 signaling for CDV immune evasion and provides a starting point for the development of morbillivirus vectors with reduced immunosuppressive properties. IMPORTANCE: The V proteins of paramyxoviruses interfere with the recognition of the virus by the immune system of the host. For morbilliviruses, the V protein is known to interact with the signal transducer and activator of transcription 1 (STAT1) and STAT2 and the melanoma differentiation-associated protein 5 (mda5), which are involved in interferon signaling. Here, we examined the contribution of each of these signaling pathways to the pathogenesis of the carnivore morbillivirus canine distemper virus. Using viruses selectively unable to interfere with the respective signaling pathway to infect ferrets, we found that inhibition of STAT2 and mda5 signaling was critical for lethal disease. Our findings provide new insights in the mechanisms of morbillivirus immune evasion and may lead to the development of new vaccines and oncolytic vectors.
Assuntos
Vírus da Cinomose Canina/patogenicidade , Cinomose/metabolismo , Interferons/metabolismo , RNA Helicases/metabolismo , Fator de Transcrição STAT2/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Núcleo Celular/metabolismo , Sequência Conservada , Cinomose/imunologia , Vírus da Cinomose Canina/genética , Vírus da Cinomose Canina/imunologia , Furões , Humanos , Masculino , Dados de Sequência Molecular , Mutação , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Ligação Proteica , Transporte Proteico , Fator de Transcrição STAT1 , Transdução de Sinais , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo , Virulência/genética , Replicação ViralRESUMO
BACKGROUND: The tumour microenvironment (TME) consists of tumour-supportive immune cells, endothelial cells, and fibroblasts. PhenoCycler, a high-plex single cell spatial biology imaging platform, is used to characterize the complexity of the TME. Researchers worldwide harvest and bank tissues from mouse models which are employed to model a plethora of human disease. With the explosion of interest in spatial biology, these panoplies of archival tissues provide a valuable resource to answer new questions. Here, we describe our protocols for developing tunable PhenoCycler multiplexed imaging panels and describe our open-source data analysis pipeline. Using these protocols, we used PhenoCycler to spatially resolve the TME of 8 routinely employed pre-clinical models of lymphoma, breast cancer, and melanoma preserved as FFPE. RESULTS: Our data reveal distinct TMEs in the different cancer models that were imaged and show that cell-cell contacts differ depending on the tumour type examined. For instance, we found that the immune infiltration in a murine model of melanoma is altered in cellular organization in melanomas that become resistant to αPD-1 therapy, with depletions in a number of cell-cell interactions. CONCLUSIONS: This work presents a valuable resource study seamlessly adaptable to any field of research involving murine models. The methodology described allows researchers to address newly formed hypotheses using archival materials, bypassing the new to perform new mouse studies.
RESUMO
Allostery plays a key role in the regulation of the activity and function of many biomolecules. And although many ligands act through allostery, no systematic use is made of it in drug design strategies. Here we describe a procedure for identifying the regions of a protein that can be used to control its activity through allostery. This procedure is based on the construction of a plausible conformational path, which describes protein transition between known active and inactive conformations. The path is calculated by using a framework approach that steers and markedly improves the conjugate peak refinement method. The evolution of conformations along this path was used to identify a putative allosteric site that could regulate activation of Bacillus anthracis adenylyl cyclase toxin (EF) by calmodulin. Conformations of the allosteric site at different steps along the path from the inactive (free) to the active (bound to calmodulin) forms of EF were used to perform virtual screenings and propose candidate EF inhibitors. Several candidates then proved to inhibit calmodulin-induced activation in an in vitro assay. The most potent compound fully inhibited EF at a concentration of 10 microM. The compounds also inhibited the related adenylyl cyclase toxin from Bordetella pertussis (CyaA). The specific homology between the putative allosteric sites in both toxins supports that these pockets are the actual binding sites of the selected inhibitors.
Assuntos
Antígenos de Bactérias/metabolismo , Bacillus anthracis/metabolismo , Toxinas Bacterianas/metabolismo , Calmodulina/química , Sítio Alostérico , Toxinas Bacterianas/antagonistas & inibidores , Bordetella pertussis/metabolismo , Química Farmacêutica/métodos , Biologia Computacional/métodos , Bases de Dados de Proteínas , Desenho de Fármacos , Humanos , Modelos Moleculares , Conformação Proteica , Estrutura Terciária de ProteínaRESUMO
Cells can rapidly adjust their proteomes in dynamic environments by regulating mRNA translation. There is mounting evidence that dysregulation of mRNA translation supports the survival and adaptation of cancer cells, which has stimulated clinical interest in targeting elements of the translation machinery and, in particular, components of the eukaryotic initiation factor 4F (eIF4F) complex such as eIF4E. However, the effect of targeting mRNA translation on infiltrating immune cells and stromal cells in the tumour microenvironment (TME) has, until recently, remained unexplored. In this Perspective article, we discuss how eIF4F-sensitive mRNA translation controls the phenotypes of key non-transformed cells in the TME, with an emphasis on the underlying therapeutic implications of targeting eIF4F in cancer. As eIF4F-targeting agents are in clinical trials, we propose that a broader understanding of their effect on gene expression in the TME will reveal unappreciated therapeutic vulnerabilities that could be used to improve the efficacy of existing cancer therapies.
Assuntos
Fator de Iniciação 4F em Eucariotos , Neoplasias , Humanos , Fator de Iniciação 4F em Eucariotos/genética , Fator de Iniciação 4F em Eucariotos/metabolismo , Microambiente Tumoral/genética , Biossíntese de Proteínas , Fator de Iniciação 4E em Eucariotos/genética , Fator de Iniciação 4E em Eucariotos/metabolismo , Neoplasias/genéticaRESUMO
Melanomas reprogram their metabolism to rapidly adapt to therapy-induced stress conditions, allowing them to persist and ultimately develop resistance. We report that a subpopulation of melanoma cells tolerate MAPK pathway inhibitors (MAPKis) through a concerted metabolic reprogramming mediated by peroxisomes and UDP-glucose ceramide glycosyltransferase (UGCG). Compromising peroxisome biogenesis, by repressing PEX3 expression, potentiated the proapoptotic effects of MAPKis via an induction of ceramides, an effect limited by UGCG-mediated ceramide metabolism. Cotargeting PEX3 and UGCG selectively eliminated a subset of metabolically active, drug-tolerant CD36+ melanoma persister cells, thereby sensitizing melanoma to MAPKis and delaying resistance. Increased levels of peroxisomal genes and UGCG were found in patient-derived MAPKi-relapsed melanomas, and simultaneously inhibiting PEX3 and UGCG restored MAPKi sensitivity in multiple models of therapy resistance. Finally, combination therapy consisting of a newly identified inhibitor of the PEX3-PEX19 interaction, a UGCG inhibitor, and MAPKis demonstrated potent antitumor activity in preclinical melanoma models, thus representing a promising approach for melanoma treatment.
Assuntos
Melanoma , Peroxissomos , Humanos , Peroxissomos/metabolismo , Metabolismo dos Lipídeos , Melanoma/genética , Ceramidas/farmacologia , Ceramidas/metabolismoRESUMO
Aberrant cell-cycle progression is characteristic of melanoma, and CDK4/6 inhibitors, such as palbociclib, are currently being tested for efficacy in this disease. Despite the promising nature of CDK4/6 inhibitors, their use as single agents in melanoma has shown limited clinical benefit. Herein, we discovered that treatment of tumor cells with palbociclib induces the phosphorylation of the mRNA translation initiation factor eIF4E. When phosphorylated, eIF4E specifically engenders the translation of mRNAs that code for proteins involved in cell survival. We hypothesized that cancer cells treated with palbociclib use upregulated phosphorylated eIF4E (phospho-eIF4E) to escape the antitumor benefits of this drug. Indeed, we found that pharmacologic or genetic disruption of MNK1/2 activity, the only known kinases for eIF4E, enhanced the ability of palbociclib to decrease clonogenic outgrowth. Moreover, a quantitative proteomics analysis of melanoma cells treated with combined MNK1/2 and CDK4/6 inhibitors showed downregulation of proteins with critical roles in cell-cycle progression and mitosis, including AURKB, TPX2, and survivin. We also observed that palbociclib-resistant breast cancer cells have higher basal levels of phospho-eIF4E, and that treatment with MNK1/2 inhibitors sensitized these palbociclib-resistant cells to CDK4/6 inhibition. In vivo we demonstrate that the combination of MNK1/2 and CDK4/6 inhibition significantly increases the overall survival of mice compared with either monotherapy. Overall, our data support MNK1/2 inhibitors as promising drugs to potentiate the antineoplastic effects of palbociclib and overcome therapy-resistant disease.
Assuntos
Neoplasias da Mama , Melanoma , Inibidores de Proteínas Quinases , Animais , Camundongos , Fator de Iniciação 4E em Eucariotos , Melanoma/tratamento farmacológico , Piperazinas/farmacologia , Piridinas/farmacologia , Neoplasias da Mama/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Antineoplásicos/farmacologiaRESUMO
The extracellular matrix (ECM) plays critical roles in breast cancer development. Whether ECM composition is regulated by the phosphorylation of eIF4E on serine 209, an event required for tumorigenesis, has not been explored. Herein, we used proteomics and mouse modeling to investigate the impact of mutating serine 209 to alanine on eIF4E (i.e., S209A) on mammary gland (MG) ECM. The proteomic data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD028953. We discovered that S209A knock-in mice, expressing a non-phosphorylatable form of eIF4E, have less collagen-I deposition in native and tumor-bearing MGs, leading to altered tumor cell invasion. Additionally, phospho-eIF4E deficiency impacts collagen topology; fibers at the tumor-stroma boundary in phospho-eIF4E-deficient mice run parallel to the tumor edge but radiate outwards in wild-type mice. Finally, a phospho-eIF4E-deficient tumor microenvironment resists anti-PD-1 therapy-induced collagen deposition, correlating with an increased anti-tumor response to immunotherapy. Clinically, we showed that collagen-I and phospho-eIF4E are positively correlated in human breast cancer samples, and that stromal phospho-eIF4E expression is influenced by tumor proximity. Together, our work defines the importance of phosphorylation of eIF4E on S209 as a regulator of MG collagen architecture in the tumor microenvironment, thereby positioning phospho-eIF4E as a therapeutic target to augment response to therapy.
Assuntos
Neoplasias da Mama , Glândulas Mamárias Humanas , Animais , Neoplasias da Mama/metabolismo , Colágeno/metabolismo , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Fator de Iniciação 4E em Eucariotos/metabolismo , Feminino , Humanos , Glândulas Mamárias Humanas/metabolismo , Camundongos , Fosforilação , Proteômica , Serina/metabolismo , Microambiente TumoralRESUMO
Promoting the macroautophagy/autophagy-mediated degradation of specific proteins and organelles can potentially be utilized to induce apoptosis in cancer cells or sensitize tumor cells to therapy. To examine this concept, we enriched for autophagosomes from histone deacetylase inhibitor (HDACi)-sensitive U937 lymphoma cells and isogenic HDACi-resistant cells. Mass spectrometry on autophagosome-enriched fractions revealed that HDACi-resistant cells undergo elevated pexophagy, or autophagy of the peroxisome, an organelle that supports tumor growth. To disturb peroxisome homeostasis, we enhanced pexophagy in HDACi-resistant cells via genetic silencing of peroxisome exportomer complex components (PEX1, PEX6, or PEX26). This consequently sensitized resistant cells to HDACi-mediated apoptosis, which was rescued by inhibiting ATM/ataxia-telangiectasia mutated (ATM serine/threonine kinase), a mediator of pexophagy. We subsequently engineered melanoma cells to stably repress PEX26 using CRISPR interference (CRISPRi). Melanoma cells with repressed PEX26 expression showed evidence of both increased pexophagy and peroxisomal matrix protein import defects versus single guide scrambled (sgSCR) controls. In vivo studies showed that sgPEX26 melanoma xenografts recurred less compared to sgSCR xenografts, following the development of resistance to mitogen-activated protein kinase (MAPK)-targeted therapy. Finally, prognostic analysis of publicly available datasets showed that low expression levels of PEX26, PEX6 and MTOR, were significantly associated with prolonged patient survival in lymphoma, lung cancer and melanoma cohorts. Our work highlighted that drugs designed to disrupt peroxisome homeostasis may serve as unconventional therapies to combat therapy resistance in cancer.Abbreviations: ABCD3/PMP70: ATP binding cassette subfamily D member 3; ACOX1: acyl-CoA oxidase 1; AP: autophagosome; COX: cytochrome c oxidase; CQ: chloroquine; CRISPRi: clustered regularly interspaced short palindromic repeats interference; DLBCL: diffuse large B-cell lymphoma; GO: gene ontology; dCas9: Cas9 endonuclease dead, or dead Cas9; HDACi: histone deacetylase inhibitors; IHC: Immunohistochemistry; LAMP2: lysosomal associated membrane protein 2; LCFAs: long-chain fatty acids; LFQ-MS: label-free quantitation mass spectrometry; LPC: lysophoshatidylcholine; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; PBD: peroxisome biogenesis disorders; PTS1: peroxisomal targeting signal 1; ROS: reactive oxygen species; sgRNA: single guide RNA; VLCFAs: very-long chain fatty acids; Vor: vorinostat; WO: wash-off.
Assuntos
Autofagia , Melanoma , ATPases Associadas a Diversas Atividades Celulares/genética , Autofagia/genética , Resistência a Medicamentos , Ácidos Graxos/metabolismo , Humanos , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/metabolismo , Proteínas de Membrana/metabolismo , Peroxissomos/metabolismo , Serina-Treonina Quinases TOR/metabolismoRESUMO
While isolating immune cells from spleens and lungs is routinely achieved using flow cytometry, it is challenging to isolate viable immune cells from skin. Here, we describe a step-by-step protocol for skin digestion using a murine melanoma model, which is amenable for detection of low abundant immune cell populations including group 2 innate lymphoid cells.
Assuntos
Imunofenotipagem , Melanoma Experimental/imunologia , Neoplasias Cutâneas/imunologia , Pele/imunologia , Animais , Citometria de Fluxo/métodos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
To determine causes of apoptotic resistance, we analyzed 124 primary B cell NHL samples using BH3 profiling, a technique that measures the mitochondrial permeabilization upon exposure to synthetic BH3 peptides. Our cohort included samples from chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), high-grade B cell lymphoma with translocations in MYC and BCL2 (HGBL-DH), mantle cell lymphoma (MCL) and marginal zone lymphoma (MZL). While a large number of our samples displayed appropriate responses to apoptosis-inducing peptides, pro-apoptotic functional defects, implicating BAX, BAK, BIM or BID, were seen in 32.4% of high-grade NHLs (12/37) and in 3.4% of low-grade NHLs (3/87, p < 0.0001). The inhibition of single anti-apoptotic proteins induced apoptosis in only a few samples, however, the dual inhibition of BCL2 and MCL1 was effective in 83% of samples, indicating MCL1 was the most common cause of lack of response to the BCL2 inhibitor, venetoclax. We then profiled Toledo and OCI-Ly8 high-grade lymphoma cell lines to determine which drugs could reduce MCL1 expression and potentiate venetoclax responses. Doxorubicin and vincristine decreased levels of MCL1 and increased venetoclax-induced apoptosis (all p < 0.05). Overall, in primary NHLs expressing BCL2 that have no defects in pro-apoptotic signaling, a poor response to venetoclax is primarily due to the presence of MCL1, which may be overcome by combining venetoclax with doxorubicin and vincristine-based chemotherapy or with other anti-microtubule inhibitors.
RESUMO
Breast cancer diagnosed within 10 years following childbirth is defined as postpartum breast cancer (PPBC) and is highly metastatic. Interactions between immune cells and other stromal cells within the involuting mammary gland are fundamental in facilitating an aggressive tumor phenotype. The MNK1/2-eIF4E axis promotes translation of prometastatic mRNAs in tumor cells, but its role in modulating the function of nontumor cells in the PPBC microenvironment has not been explored. Here, we used a combination of in vivo PPBC models and in vitro assays to study the effects of inactivation of the MNK1/2-eIF4E axis on the protumor function of select cells of the tumor microenvironment. PPBC mice deficient for phospho-eIF4E (eIF4ES209A) were protected against lung metastasis and exhibited differences in the tumor and lung immune microenvironment compared with wild-type mice. Moreover, the expression of fibroblast-derived IL33, an alarmin known to induce invasion, was repressed upon MNK1/2-eIF4E axis inhibition. Imaging mass cytometry on PPBC and non-PPBC patient samples indicated that human PPBC contains phospho-eIF4E high-expressing tumor cells and CD8+ T cells displaying markers of an activated dysfunctional phenotype. Finally, inhibition of MNK1/2 combined with anti-PD-1 therapy blocked lung metastasis of PPBC. These findings implicate the involvement of the MNK1/2-eIF4E axis during PPBC metastasis and suggest a promising immunomodulatory route to enhance the efficacy of immunotherapy by blocking phospho-eIF4E. SIGNIFICANCE: This study investigates the MNK1/2-eIF4E signaling axis in tumor and stromal cells in metastatic breast cancer and reveals that MNK1/2 inhibition suppresses metastasis and sensitizes tumors to anti-PD-1 immunotherapy.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Fator de Iniciação 4E em Eucariotos/uso terapêutico , Terapia de Imunossupressão/métodos , Animais , Modelos Animais de Doenças , Fator de Iniciação 4E em Eucariotos/farmacologia , Feminino , Humanos , Camundongos , Metástase Neoplásica , Período Pós-PartoRESUMO
Melanomas commonly undergo a phenotype switch, from a proliferative to an invasive state. Such tumor cell plasticity contributes to immunotherapy resistance; however, the mechanisms are not completely understood and thus are therapeutically unexploited. Using melanoma mouse models, we demonstrated that blocking the MNK1/2-eIF4E axis inhibited melanoma phenotype switching and sensitized melanoma to anti-PD-1 immunotherapy. We showed that phospho-eIF4E-deficient murine melanomas expressed high levels of melanocytic antigens, with similar results verified in patient melanomas. Mechanistically, we identified phospho-eIF4E-mediated translational control of NGFR, a critical effector of phenotype switching. Genetic ablation of phospho-eIF4E reprogrammed the immunosuppressive microenvironment, exemplified by lowered production of inflammatory factors, decreased PD-L1 expression on dendritic cells and myeloid-derived suppressor cells, and increased CD8+ T cell infiltrates. Finally, dual blockade of the MNK1/2-eIF4E axis and the PD-1/PD-L1 immune checkpoint demonstrated efficacy in multiple melanoma models regardless of their genomic classification. An increase in the presence of intratumoral stem-like TCF1+PD-1+CD8+ T cells, a characteristic essential for durable antitumor immunity, was detected in mice given a MNK1/2 inhibitor and anti-PD-1 therapy. Using MNK1/2 inhibitors to repress phospho-eIF4E thus offers a strategy to inhibit melanoma plasticity and improve response to anti-PD-1 immunotherapy.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Fator de Iniciação 4E em Eucariotos/imunologia , Imunidade Celular , Sistema de Sinalização das MAP Quinases/imunologia , Melanoma Experimental/imunologia , Proteínas Serina-Treonina Quinases/imunologia , Animais , Antígeno B7-H1/genética , Antígeno B7-H1/imunologia , Linhagem Celular Tumoral , Fator de Iniciação 4E em Eucariotos/genética , Imunoterapia , Sistema de Sinalização das MAP Quinases/genética , Melanoma Experimental/genética , Melanoma Experimental/terapia , Camundongos , Camundongos Transgênicos , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/imunologia , Proteínas Serina-Treonina Quinases/genética , Receptor de Fator de Crescimento Neural/genética , Receptor de Fator de Crescimento Neural/imunologiaRESUMO
The sensory characteristics of white and milk chocolate with three origins of vanilla (Madagascar, Indonesia, Papua New Guinea) were investigated using a multi-analytical approach. The sensory tests included profiling using Quantitative Descriptive Analysis with a trained panel; Temporal Dominance of Sensations test with untrained respondents from various countries currently residing in Belgium, and the consumers sensory preference test with consumers in Belgium, France, Sweden and Canada. The vanilla extracts were also analyzed by gas chromatography-mass spectrometry (GC-MS) in combination with olfactometry (GC-MS-O) to identify the key aroma compounds in the different vanilla origins. This research shows that sensory profile of Papua New Guinea (PNG) samples is different from Madagascan and Indonesian extracts. The flavor signature was correlated with almondy and anisic descriptors. The perceived difference between Madagascan and Indonesian origins was reduced when assessed within complex matrices such as chocolate. In sugared water, Madagascan vanilla has balsamic, phenolic notes while Indonesian has woodier notes. In a chocolate base, the Indonesian sample lost its woody characteristic for a dairy enhanced characteristic. The botanical variety has more impact on difference in sensory profile of a chocolate than the vanilla origin itself. The sensory data correlates with volatile components identified in this study. Guaiacol and vanillin were identified as the main key aroma compounds in all three vanilla origins using GC-MS-O. Additional key aroma compounds were responsible for the differences in sensory characteristics between the three vanilla origins: anisaldehyde for anisic note in the PNG samples and ethyl 2-methylbutanoate and 3/2-methylbutanoic acid in the Indonesian sample. Sensory profiles of vanillas from various origins were more visible and pronounced in white chocolate compared to milk chocolate. This study gives novel insights to the chocolate, as well as the vanilla sectors, indicating possibilities of vanilla replacement and consumer preferences across Europe and Canada. The consumer study shows that, on average, people do not indicate which chocolate is significantly more liked.
Assuntos
Chocolate , Vanilla , Bélgica , Canadá , Europa (Continente) , França , Humanos , Indonésia , MadagáscarRESUMO
The BRAFV600E mutation occurs in more than 50% of cutaneous melanomas, and results in the constitutive activation of the mitogen-activated protein kinases (MAPK) pathway. MAP kinase-interacting serine/threonine-protein kinase 1 and 2 (MNK1/2) are downstream effectors of the activated MAPK pathway, and important molecular targets in invasive and metastatic cancer. Despite the well-known role of MNK1 in regulating mRNA translation, little is known concerning the impact of its aberrant activation on gene transcription. Here, we show that changes in the activity, or abundance, of MNK1 result in changes in the expression of pro-oncogenic and pro-invasive genes. Among the MNK1-upregulated genes, we identify Angiopoietin-like 4 (ANGPTL4), which in turn promotes an invasive phenotype via its ability to induce the expression of matrix metalloproteinases (MMPs). Using a pharmacologic inhibitor of MNK1/2, SEL201, we demonstrate that BRAFV600E-mutated cutaneous melanoma cells are reliant on MNK1/2 for invasion and lung metastasis.
Assuntos
Proteína 4 Semelhante a Angiopoietina/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias Pulmonares/genética , Melanoma/tratamento farmacológico , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas B-raf/genética , Neoplasias Cutâneas/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Progressão da Doença , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Inativação de Genes , Xenoenxertos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Melanoma/genética , Melanoma/patologia , Camundongos , Mutação/genética , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Transcriptoma/genética , Melanoma Maligno CutâneoRESUMO
The tremendous success of immune checkpoint blockades has revolutionized cancer management. Our increased understanding of the cell types that compose the tumor microenvironment (TME), including those of the innate and adaptive immune system, has helped to shape additional immune modulatory strategies in cancer care. Pre-clinical and clinical investigations targeting novel checkpoint interactions and key pathways that regulate cancer immunity continue to increase rapidly. Various combinatorial drug regimens are being tested in attempt to achieve durable response and survival rates of patients with cancer. This review provides an overview of specific components of the TME, an introduction to novel immune checkpoints, followed by a survey of present day and future combination immune modulatory therapies. The idea that the immune system can recognize and destroy tumor cells was first described in the cancer immunosurveillance hypothesis of Burnet and Thomas. However, early experimental evidence failed to support the concept. It was not until the late 1990s when seminal papers clearly showed the existence of cancer immunosurveillance, leading to the cancer immunoediting hypothesis. In this century, progress in the understanding of negative regulators of the immune response led to the discovery that inhibition of these regulators in patients with cancer could lead to dramatic and durable remissions. Drs. Tasuku Honjo and James P. Allison were awarded the Nobel Prize in 2018 for their pioneering work in this field. We now see rapid advances in cancer immunology and emerging effective therapies revolutionizing cancer care across tumor types in the clinic, while pre-clinical research is moving from a focus on the malignant cells themselves to dissect the highly heterogenic and complex multi-cellular tumor microenvironment (TME).
Assuntos
Imunoterapia/métodos , Neoplasias/terapia , Microambiente Tumoral/imunologia , Animais , Distinções e Prêmios , Humanos , Neoplasias/imunologia , Prêmio Nobel , Pesquisa Translacional BiomédicaRESUMO
The mechanisms by which breast cancers progress from relatively indolent ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) are not well understood. However, this process is critical to the acquisition of metastatic potential. MAPK-interacting serine/threonine-protein kinase 1 (MNK1) signaling can promote cell invasion. NODAL, a morphogen essential for embryogenic patterning, is often reexpressed in breast cancer. Here we describe a MNK1/NODAL signaling axis that promotes DCIS progression to IDC. We generated MNK1 knockout (KO) or constitutively active MNK1 (caMNK1)-expressing human MCF-10A-derived DCIS cell lines, which were orthotopically injected into the mammary glands of mice. Loss of MNK1 repressed NODAL expression, inhibited DCIS to IDC conversion, and decreased tumor relapse and metastasis. Conversely, caMNK1 induced NODAL expression and promoted IDC. The MNK1/NODAL axis promoted cancer stem cell properties and invasion in vitro. The MNK1/2 inhibitor SEL201 blocked DCIS progression to invasive disease in vivo. In clinical samples, IDC and DCIS with microinvasion expressed higher levels of phospho-MNK1 and NODAL versus low-grade (invasion-free) DCIS. Cumulatively, our data support further development of MNK1 inhibitors as therapeutics for preventing invasive disease. SIGNIFICANCE: These findings provide new mechanistic insight into progression of ductal carcinoma and support clinical application of MNK1 inhibitors to delay progression of indolent ductal carcinoma in situ to invasive ductal carcinoma.