RESUMO
Therapy of BRAF-mutant melanoma with selective inhibitors of BRAF (BRAFi) and MEK (MEKi) represents a major clinical advance but acquired resistance to therapy has emerged as a key obstacle. To date, no clinical approaches successfully resensitize to BRAF/MEK inhibition. Here, we develop a therapeutic strategy for melanoma using bromosporine, a bromodomain inhibitor. Bromosporine (bromo) monotherapy produced significant anti-tumor effects against established melanoma cell lines and patient-derived xenografts (PDXs). Combinatorial therapy involving bromosporine and cobimetinib (bromo/cobi) showed synergistic anti-tumor effects in multiple BRAFi-resistant PDX models. The bromo/cobi combination was superior in vivo to standard BRAFi/MEKi therapy in the treatment-naive BRAF-mutant setting and to MEKi alone in the setting of immunotherapy-resistant NRAS- and NF1-mutant melanoma. RNA sequencing of xenografts treated with bromo/cobi revealed profound down-regulation of genes critical to cell division and mitotic progression. Bromo/cobi treatment resulted in marked DNA damage and cell-cycle arrest, resulting in induction of apoptosis. These studies introduce bromodomain inhibition, alone or combined with agents targeting the mitogen activated protein kinase pathway, as a rational therapeutic approach for melanoma refractory to standard targeted or immunotherapeutic approaches.
Assuntos
Melanoma , Proteínas Proto-Oncogênicas B-raf , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/patologia , Quinases de Proteína Quinase Ativadas por Mitógeno , Proteínas Nucleares , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas B-raf/metabolismo , Fatores de TranscriçãoRESUMO
BACKGROUND: While melanomas commonly harbor losses of 9p21, on which CDKN2A resides, the presence of additional tumor suppressor elements at this locus is incompletely characterized. Here we assess the expression levels and functional role of microRNA-876-3p (miR-876), whose gene also maps to 9p21. METHODS: Expression of miR-876 was assessed in human tissues and cell lines using quantitative miRNA reverse transcriptase polymerase chain reaction (qRT-PCR). MIR876 copy number was determined in The Cancer Genome Atlas (TCGA) melanoma cohort. The consequences of regulation of miR-876 expression were assessed on melanoma cell colony formation, migration, invasion, apoptosis, cell cycle progression, and drug sensitivity in culture, and on in vivo tumor growth in a xenograft model. Genome-wide transcriptomic changes induced by miR-876 overexpression were determined using RNA sequencing (RNA-Seq). RESULTS: miR-876 expression was significantly decreased in primary melanoma samples when compared with nevi, and in human melanoma cell lines when compared with human melanocytes. Analysis of the TCGA cohort revealed deletions in MIR876 in > 50% of melanomas. miR-876 overexpression resulted in decreased melanoma cell colony formation, migration, and invasion, which was accompanied by cell cycle arrest and increased apoptosis. Intra-tumoral injections of miR-876 significantly suppressed melanoma growth in vivo. RNA-Seq analysis of miR-876-treated tumors revealed downregulation of several growth-promoting genes, along with upregulation of tumor suppressor genes, which was confirmed by qRT-PCR analysis. Computational analyses identified MAPK1 (or ERK2) as a possible target of miR-876 action. Overexpression of miR-876 significantly suppressed luciferase expression driven by the MAPK1/ERK2 3' UTR, and resulted in decreased ERK protein expression in melanoma cells. MAPK1/ERK2 cDNA overexpression rescued the effects of miR-876 on melanoma colony formation. miR-876 overexpression sensitized melanoma cells to treatment with the BRAF inhibitor vemurafenib. CONCLUSIONS: These studies identify miR-876 as a distinct tumor suppressor on 9p21 that is inactivated in melanoma and suggest miR-876 loss as an additional mechanism to activate ERK and the mitogen activated protein kinase (MAPK) pathway in melanoma. In addition, they suggest the therapeutic potential of combining miR-876 overexpression with BRAF inhibition as a rational therapeutic strategy for melanoma.
Assuntos
Cromossomos Humanos Par 9 , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Melanoma , MicroRNAs , Humanos , Melanoma/genética , Melanoma/patologia , MicroRNAs/genética , MicroRNAs/metabolismo , Linhagem Celular Tumoral , Animais , Cromossomos Humanos Par 9/genética , Movimento Celular/genética , Apoptose/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proliferação de Células/genética , Sequência de Bases , Invasividade NeoplásicaRESUMO
The invasive behavior of glioblastoma is essential to its aggressive potential. Here, we show that pleckstrin homology domain interacting protein (PHIP), acting through effects on the force transduction layer of the focal adhesion complex, drives glioblastoma motility and invasion. Immunofluorescence analysis localized PHIP to the leading edge of glioblastoma cells, together with several focal adhesion proteins: vinculin (VCL), talin 1 (TLN1), integrin beta 1 (ITGB1), as well as phosphorylated forms of paxillin (pPXN) and focal adhesion kinase (pFAK). Confocal microscopy specifically localized PHIP to the force transduction layer, together with TLN1 and VCL. Immunoprecipitation revealed a physical interaction between PHIP and VCL. Targeted suppression of PHIP resulted in significant down-regulation of these focal adhesion proteins, along with zyxin (ZYX), and produced profoundly disorganized stress fibers. Live-cell imaging of glioblastoma cells overexpressing a ZYX-GFP construct demonstrated a role for PHIP in regulating focal adhesion dynamics. PHIP silencing significantly suppressed the migratory and invasive capacity of glioblastoma cells, partially restored following TLN1 or ZYX cDNA overexpression. PHIP knockdown produced substantial suppression of tumor growth upon intracranial implantation, as well as significantly reduced microvessel density and secreted VEGF levels. PHIP copy number was elevated in the classical glioblastoma subtype and correlated with elevated EGFR levels. These results demonstrate PHIP's role in regulating the actin cytoskeleton, focal adhesion dynamics, and tumor cell motility, and identify PHIP as a key driver of glioblastoma migration and invasion.
Assuntos
Neoplasias Encefálicas/patologia , Adesões Focais/patologia , Glioblastoma/patologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neovascularização Patológica/patologia , Citoesqueleto de Actina/metabolismo , Animais , Encéfalo/patologia , Neoplasias Encefálicas/irrigação sanguínea , Neoplasias Encefálicas/genética , Adesão Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Estudos de Coortes , Progressão da Doença , Feminino , Dosagem de Genes , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Glioblastoma/irrigação sanguínea , Glioblastoma/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Microscopia Intravital , Camundongos , Microscopia Confocal , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Neovascularização Patológica/genética , Imagem com Lapso de Tempo , Vinculina/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The identification and targeting of key molecular drivers of melanoma and breast and lung cancer have substantially improved their therapy. However, subtypes of each of these three common, lethal solid tumors lack identified molecular drivers, and are thus not amenable to targeted therapies. Here we show that pleckstrin homology domain-interacting protein (PHIP) promotes the progression of these "driver-negative" tumors. Suppression of PHIP expression significantly inhibited both tumor cell proliferation and invasion, coordinately suppressing phosphorylated AKT, cyclin D1, and talin1 expression in all three tumor types. Furthermore, PHIP's targetable bromodomain is functional, as it specifically binds the histone modification H4K91ac. Analysis of TCGA profiling efforts revealed PHIP overexpression in triple-negative and basal-like breast cancer, as well as in the bronchioid subtype of nonsmall cell lung cancer. These results identify a role for PHIP in the progression of melanoma and breast and lung cancer subtypes lacking identified targeted therapies. The use of selective, anti-PHIP bromodomain inhibitors may thus yield a broad-based, molecularly targeted therapy against currently nontargetable tumors.
Assuntos
Mama/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Pulmonares/metabolismo , Melanoma/metabolismo , Domínios de Homologia à Plecstrina/fisiologia , Neoplasias de Mama Triplo Negativas/metabolismo , Animais , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Ciclina D1/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
BACKGROUND: Patient-derived xenograft (PDX) mouse tumour models can predict response to therapy in patients. Predictions made from PDX cultures (PDXC) would allow for more rapid and comprehensive evaluation of potential treatment options for patients, including drug combinations. METHODS: We developed a PDX library of BRAF-mutant metastatic melanoma, and a high-throughput drug-screening (HTDS) platform utilising clinically relevant drug exposures. We then evaluated 34 antitumor agents across eight melanoma PDXCs, compared drug response to BRAF and MEK inhibitors alone or in combination with PDXC and the corresponding PDX, and investigated novel drug combinations targeting BRAF inhibitor-resistant melanoma. RESULTS: The concordance of cancer-driving mutations across patient, matched PDX and subsequent PDX generations increases as variant allele frequency (VAF) increases. There was a high correlation in the magnitude of response to BRAF and MEK inhibitors between PDXCs and corresponding PDXs. PDXCs and corresponding PDXs from metastatic melanoma patients that progressed on standard-of-care therapy demonstrated similar resistance patterns to BRAF and MEK inhibitor therapy. Importantly, HTDS identified novel drug combinations to target BRAF-resistant melanoma. CONCLUSIONS: The biological consistency observed between PDXCs and PDXs suggests that PDXCs may allow for a rapid and comprehensive identification of treatments for aggressive cancers, including combination therapies.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Melanoma/tratamento farmacológico , Animais , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , MAP Quinase Quinase Quinases/antagonistas & inibidores , Melanoma/enzimologia , Melanoma/genética , Melanoma/patologia , Camundongos , Mutação , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Distribuição Aleatória , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Cellular senescence is an essentially irreversible arrest of cell proliferation coupled to a complex senescence-associated secretory phenotype (SASP). The senescence arrest prevents the development of cancer, and the SASP can promote tissue repair. Recent data suggest that the prolonged presence of senescent cells, and especially the SASP, could be deleterious, and their beneficial effects early in life can become maladaptive such that they drive aging phenotypes and pathologies late in life. It is therefore important to develop strategies to eliminate senescent cells. There are currently under development or approved several immune cell-based therapies for cancer, which could be redesigned to target senescent cells. This review focuses on this possible use of immune cells and discusses how current cell-based therapies could be used for senescent cell removal.
RESUMO
Inhibitors of DNA-binding (ID) proteins are negative regulators of basic helix-loop-helix transcription factors and generally stimulate cell proliferation and inhibit differentiation. We previously determined that ID1 was highly expressed in aggressive salivary gland cancer (SGC) cells in culture. Here, we show that ID2 is also expressed in aggressive SGC cells. ID2 knockdown triggers important changes in cell behavior, that is, it significantly reduces the expression of N-cadherin, vimentin and Snail, induces E-cadherin expression and leads to a more differentiated phenotype exemplified by changes in cell shape. Moreover, ID2 knockdown almost completely suppresses invasion and the expression of matrix metalloproteinase 9. In conclusion, ID2 expression maintains an aggressive phenotype in SGC cells, and ID2 repression triggers a reduction in cell aggressiveness. ID2 therefore represents a potential therapeutic target during SGC progression. ID proteins are negative regulators of basic helix-loop-helix transcription factors and generally stimulate cell proliferation and inhibit differentiation. ID2 knockdown triggers important changes in cell behavior, that is, it significantly reduces the expression of N-cadherin, vimentin and Snail, induces E-cadherin expression and leads to a more differentiated phenotype exemplified by changes in cell shape. ID2 therefore represents a potential therapeutic target during SGC progression.
Assuntos
Proliferação de Células/genética , Proteína 2 Inibidora de Diferenciação/genética , Terapia de Alvo Molecular , Neoplasias das Glândulas Salivares/genética , Caderinas/biossíntese , Diferenciação Celular/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Proteína 2 Inibidora de Diferenciação/biossíntese , Invasividade Neoplásica/genética , Fenótipo , Neoplasias das Glândulas Salivares/patologia , Neoplasias das Glândulas Salivares/terapia , Fatores de Transcrição da Família Snail/biossíntese , Vimentina/biossínteseRESUMO
Cellular senescence is an established tumour-suppressive mechanism that prevents the proliferation of premalignant cells. However, several lines of evidence show that senescent cells, which often persist in vivo, can also promote tumour progression in addition to other age-related pathologies via the senescence-associated secretory phenotype (SASP). Moreover, new insights suggest the SASP can facilitate tissue repair. Here, we review the beneficial and detrimental roles of senescent cells, highlighting conditions under which the senescence response does and does not promote pathology, particularly cancer. By better understanding the context-dependent effects of cellular senescence, it may be feasible to limit its detrimental properties while preserving its beneficial effects, and develop novel therapeutic strategies to prevent or treat cancer and possibly other age-associated diseases.
Assuntos
Senescência Celular/fisiologia , Neoplasias/patologia , Animais , Divisão Celular , Microambiente Celular , Desenvolvimento Embrionário , Matriz Extracelular/metabolismo , Humanos , Inflamação , Camundongos , Invasividade Neoplásica , Neovascularização Patológica/fisiopatologia , Neovascularização Fisiológica , Fenótipo , RegeneraçãoRESUMO
Cellular senescence is a major driver of aging and age-related diseases. Quantification of senescent cells remains challenging due to the lack of senescence-specific markers and generalist, unbiased methodology. Here, we describe the Fully-Automated Senescence Test (FAST), an image-based method for the high-throughput, single-cell assessment of senescence in cultured cells. FAST quantifies three of the most widely adopted senescence-associated markers for each cell imaged: senescence-associated ß-galactosidase activity (SA-ß-Gal) using X-Gal, proliferation arrest via lack of 5-ethynyl-2'-deoxyuridine (EdU) incorporation, and enlarged morphology via increased nuclear area. The presented workflow entails microplate image acquisition, image processing, data analysis, and graphing. Standardization was achieved by i) quantifying colorimetric SA-ß-Gal via optical density; ii) implementing staining background controls; iii) automating image acquisition, image processing, and data analysis. In addition to the automated threshold-based scoring, a multivariate machine learning approach is provided. We show that FAST accurately quantifies senescence burden and is agnostic to cell type and microscope setup. Moreover, it effectively mitigates false-positive senescence marker staining, a common issue arising from culturing conditions. Using FAST, we compared X-Gal with fluorescent C12FDG live-cell SA-ß-Gal staining on the single-cell level. We observed only a modest correlation between the two, indicating that those stains are not trivially interchangeable. Finally, we provide proof of concept that our method is suitable for screening compounds that modify senescence burden. This method will be broadly useful to the aging field by enabling rapid, unbiased, and user-friendly quantification of senescence burden in culture, as well as facilitating large-scale experiments that were previously impractical.
RESUMO
Cellular senescence is a major driver of aging and age-related diseases. Quantification of senescent cells remains challenging due to the lack of senescence-specific markers and generalist, unbiased methodology. Here, we describe the Fully-Automated Senescence Test (FAST), an image-based method for the high-throughput, single-cell assessment of senescence in cultured cells. FAST quantifies three of the most widely adopted senescence-associated markers for each cell imaged: senescence-associated ß-galactosidase activity (SA-ß-Gal) using X-Gal, proliferation arrest via lack of 5-ethynyl-2'-deoxyuridine (EdU) incorporation, and enlarged morphology via increased nuclear area. The presented workflow entails microplate image acquisition, image processing, data analysis, and graphing. Standardization was achieved by (i) quantifying colorimetric SA-ß-Gal via optical density; (ii) implementing staining background controls; and (iii) automating image acquisition, image processing, and data analysis. In addition to the automated threshold-based scoring, a multivariate machine learning approach is provided. We show that FAST accurately quantifies senescence burden and is agnostic to cell type and microscope setup. Moreover, it effectively mitigates false-positive senescence marker staining, a common issue arising from culturing conditions. Using FAST, we compared X-Gal with fluorescent C12FDG live-cell SA-ß-Gal staining on the single-cell level. We observed only a modest correlation between the two, indicating that those stains are not trivially interchangeable. Finally, we provide proof of concept that our method is suitable for screening compounds that modify senescence burden. This method will be broadly useful to the aging field by enabling rapid, unbiased, and user-friendly quantification of senescence burden in culture, as well as facilitating large-scale experiments that were previously impractical.
Assuntos
Biomarcadores , Senescência Celular , Ensaios de Triagem em Larga Escala , beta-Galactosidase , Senescência Celular/fisiologia , Humanos , Biomarcadores/metabolismo , Ensaios de Triagem em Larga Escala/métodos , beta-Galactosidase/metabolismo , Células Cultivadas , Processamento de Imagem Assistida por Computador , Fibroblastos , Análise de Célula Única/métodosRESUMO
Electrolyzed-reduced water has powerful antioxidant properties with constituents that scavenge reactive oxygen species (ROS), which are known to be produced by several intrinsic and extrinsic processes. When there is an imbalance between ROS production and antioxidant defenses, oxidative stress occurs. Persistent oxidative stress leads to cellular senescence, an important hallmark of aging, and is involved in several age-related conditions and illnesses. This study aims to investigate whether Weo electrolyzed water (WEW) could modulate the phenotype of senescent cells. We compared normal human lung fibroblasts (BJ) and breast cancer cells (T47D) treated with hydrogen peroxide (H2O2) to induce senescence. We assessed the molecular impact of WEW on markers of cellular senescence, senescence-associated secretory phenotype (SASP) factors, and stress response genes. Treatment with WEW modulated markers of cellular senescence, such as the senescence-associated ß-galactosidase (SA-ß-gal) activity, EdU incorporation and p21 expression, similarly in both cell types. However, WEW modulated the expression of SASP factors and stress response genes in a cell type-dependent and opposite fashion, significantly decreasing them in BJ cells, while stimulating their expression in T47D cells. Reduction in the expression of SASP factors and stress-related genes in BJ cells suggests that WEW acts as a protective factor, thereby reducing oxidative stress in normal cells, while making cancer cells more sensitive to the effects of cellular stress, thus increasing their elimination and consequently reducing their deleterious effects. These findings suggest that, due to its differential effects as a senomorphic factor, WEW could have a positive impact on longevity and age-related diseases.
Assuntos
Senescência Celular , Peróxido de Hidrogênio , Estresse Oxidativo , Água , Humanos , Senescência Celular/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Linhagem Celular Tumoral , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fenótipo Secretor Associado à Senescência/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Feminino , EletróliseRESUMO
Cellular senescence has been strongly linked to aging and age-related diseases. It is well established that the phenotype of senescent cells is highly heterogeneous and influenced by their cell type and senescence-inducing stimulus. Recent single-cell RNA-sequencing studies identified heterogeneity within senescent cell populations. However, proof of functional differences between such subpopulations is lacking. To identify functionally distinct senescent cell subpopulations, we employed high-content image analysis to measure senescence marker expression in primary human endothelial cells and fibroblasts. We found that G2-arrested senescent cells feature higher senescence marker expression than G1-arrested senescent cells. To investigate functional differences, we compared IL-6 secretion and response to ABT263 senolytic treatment in G1 and G2 senescent cells. We determined that G2-arrested senescent cells secrete more IL-6 and are more sensitive to ABT263 than G1-arrested cells. We hypothesize that cell cycle dependent DNA content is a key contributor to the heterogeneity within senescent cell populations. This study demonstrates the existence of functionally distinct senescent subpopulations even in culture. This data provides the first evidence of selective cell response to senolytic treatment among senescent cell subpopulations. Overall, this study emphasizes the importance of considering the senescent cell heterogeneity in the development of future senolytic therapies.
RESUMO
PURPOSE: The objective of this study was to investigate the senescent phenotypes of human corneal endothelial cells (hCEnCs) upon treatment with ultraviolet (UV)-A. METHODS: We assessed cell morphology, senescence-associated ß-galactosidase (SA-ß-gal) activity, cell proliferation and expression of senescence markers (p16 and p21) in hCEnCs exposed to UV-A radiation, and senescent hCEnCs induced by ionizing radiation (IR) were used as positive controls. We performed RNA sequencing and proteomics analyses to compare gene and protein expression profiles between UV-A- and IR-induced senescent hCEnCs, and we also compared the results to non-senescent hCEnCs. RESULTS: Cells exposed to 5 J/cm2 of UV-A or to IR exhibited typical senescent phenotypes, including enlargement, increased SA-ß-gal activity, decreased cell proliferation and elevated expression of p16 and p21. RNA-Seq analysis revealed that 83.9% of the genes significantly upregulated and 82.6% of the genes significantly downregulated in UV-A-induced senescent hCEnCs overlapped with the genes regulated in IR-induced senescent hCEnCs. Proteomics also revealed that 93.8% of the proteins significantly upregulated in UV-A-induced senescent hCEnCs overlapped with those induced by IR. In proteomics analyses, senescent hCEnCs induced by UV-A exhibited elevated expression levels of several factors part of the senescence-associated secretory phenotype. CONCLUSIONS: In this study, where senescence was induced by UV-A, a more physiological stress for hCEnCs compared to IR, we determined that UV-A modulated the expression of many genes and proteins typically altered upon IR treatment, a more conventional method of senescence induction, even though UV-A also modulated specific pathways unrelated to IR.
Assuntos
Proliferação de Células , Senescência Celular , Células Endoteliais , Raios Ultravioleta , Humanos , Senescência Celular/efeitos da radiação , Raios Ultravioleta/efeitos adversos , Proliferação de Células/efeitos da radiação , Células Endoteliais/efeitos da radiação , Células Endoteliais/metabolismo , Endotélio Corneano/efeitos da radiação , Endotélio Corneano/metabolismo , Células Cultivadas , Proteômica , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , beta-Galactosidase/metabolismo , beta-Galactosidase/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/genéticaRESUMO
Senescence emerged as a significant mechanism of aging and age-related diseases, offering an attractive target for clinical interventions. Senescent cells release a senescence-associated secretory phenotype (SASP), including exosomes that may act as signal transducers between distal tissues, propagating secondary or bystander senescence and signaling throughout the body. However, the composition of exosome SASP remains underexplored, presenting an opportunity for novel unbiased discovery. Here, we present a detailed proteomic and lipidomic analysis of exosome SASP using mass spectrometry from human plasma from young and older individuals and from tissue culture of senescent primary human lung fibroblasts. We identified ~1,300 exosome proteins released by senescent fibroblasts induced by three different senescence inducers causing most exosome proteins to be differentially regulated with senescence. In parallel, a human plasma cohort from young and old individuals revealed over 1,350 exosome proteins and 171 plasma exosome proteins were regulated when comparing old vs young individuals. Of the age-regulated plasma exosome proteins, we observed 52 exosome SASP factors that were also regulated in exosomes from the senescent fibroblasts, including serine protease inhibitors (SERPINs), Prothrombin, Coagulation factor V, Plasminogen, and Reelin. In addition, 247 lipids were identified with high confidence in all exosome samples. Following the senescence inducers, a majority of the identified phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin species increased significantly indicating cellular membrane changes. The most notable categories of significantly changed proteins were related to extracellular matrix remodeling and inflammation, both potentially detrimental pathways that can damage surrounding tissues and even induce secondary or bystander senescence. Our findings reveal mechanistic insights and potential senescence biomarkers, enabling a better approach to surveilling the senescence burden in the aging population and offering promising therapeutic targets for interventions.
RESUMO
The evolution of primary melanoma to lymph node and distant metastasis is incompletely understood. We examined the genomic diversity in melanoma progression in matched primary melanomas and lymph node and distant metastases from 17 patients. FISH analysis revealed cancer cell fractions with monotonic copy number alterations, including PHIP gain and PTEN loss, in the metastatic cascade. By contrast, the cancer cell fraction with copy number alterations for BPTF and MITF was reduced in lymph node metastases but increased in distant metastases. Separately, the cancer cell fraction with NCOA3 copy number alteration was comparable between primary tumors and lymph node metastases yet increased in distant metastases. These results suggest enrichment of the phosphoinositide 3-kinase and MITF pathways in the transition through the metastatic cascade. By contrast, next-generation sequencing analysis did not identify a consistent pattern of changes in variant allele frequency while revealing several intriguing findings, including decreased variant allele frequency in distant metastases and distinct drivers in lymph node versus distant metastases. These results provide evidence that distant melanoma metastasis does not always emanate from lymph node metastasis. These results enhance our understanding of clonal patterns of melanoma metastasis, with possible implications for targeted therapy and metastasis competency.
RESUMO
BACKGROUND: Salivary gland cancer (SGC) is one of the common malignancies of the head and neck area. It develops in the minor and major salivary glands and sometimes metastasizes to other organs, particularly to the lungs. Inhibitors of differentiation (Id) proteins are negative regulators of basic helix-loop-helix transcription factors that control malignant cell behavior and tumor aggressiveness in many tissues. In this study, our goal was to determine the potential role of Id proteins, particularly Id1, during human SGC cell progression. METHODS: We first determined the expression levels of Id1 and Id2 in four SGC cell lines: two adenocarcinoma of the salivary gland (HSG and HSY) and two adenoid cystic carcinoma (ACC2 and ACCM) cell lines. We then used constructs that expressed antisense cDNAs to Id1 or Id2 to knockdown the expression of these proteins in cell lines where they were highly expressed, and determined the effects of the knockdown on cell proliferation, migration and invasion. RESULTS: Id1 mRNA and protein were detectable in all cell lines, and expression of Id2 was variable, from absent to high. The ACC2 and ACCM cell lines expressed both Id1 and Id2, but Id1 was expressed at a higher level in the more aggressive ACCM cell line in comparison to ACC2 cells as confirmed by Id1 promoter-reporter assays. We therefore focused on the ACCM cells for the remainder of the study. We found that proliferation and invasiveness of ACCM cells were strongly reduced after Id1 knockdown whereas Id2 suppression had only a slight effect. Results of scratch and colony formation assays also confirmed that ACCM cell aggressiveness was significantly reduced upon Id1 knockdown. Finally, this knockdown resulted in reduced c-myc and enhanced cyclin-dependent kinase inhibitor p21 expression. CONCLUSIONS: These results demonstrate that Id1 plays an important role in the control of human SGC cell aggressiveness and suggest a potential role as a marker of diagnosis, prognosis and progression of SGCs. Id1 suppression could represent a novel and effective approach for the treatment of salivary gland cancer.
Assuntos
Adenocarcinoma/genética , Carcinoma Adenoide Cístico/genética , Proteína 1 Inibidora de Diferenciação/genética , Neoplasias das Glândulas Salivares/genética , Adenocarcinoma/metabolismo , Carcinoma Adenoide Cístico/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p21/genética , Regulação para Baixo , Técnicas de Silenciamento de Genes , Humanos , Proteína 1 Inibidora de Diferenciação/metabolismo , Proteína 2 Inibidora de Diferenciação/genética , Proteína 2 Inibidora de Diferenciação/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Neoplasias das Glândulas Salivares/metabolismo , Ensaio Tumoral de Célula-Tronco , Regulação para CimaRESUMO
Most patients who die from cancer succumb to treatment-refractory advanced metastatic progression. Although the early stages of tumor metastasis result in the formation of clinically silent micrometastatic foci, its later stages primarily reflect the progressive, organ-destructive growth of already advanced metastases. Early-stage metastasis is regulated by multiple factors within tumor cells as well as by the tumor microenvironment (TME). In contrast, the molecular determinants that control advanced metastatic progression remain essentially uncharacterized, precluding the development of therapies targeted against it. Here we show that the TME, functioning in part through platelet endothelial cell adhesion molecule 1 (PECAM-1), drives advanced metastatic progression and is essential for progression through its preterminal end stage. PECAM-1-KO and chimeric mice revealed that its metastasis-promoting effects are mediated specifically through vascular endothelial cell (VEC) PECAM-1. Anti-PECAM-1 mAb therapy suppresses both end-stage metastatic progression and tumor-induced cachexia in tumor-bearing mice. It reduces proliferation, but not angiogenesis or apoptosis, within advanced tumor metastases. Because its antimetastatic effects are mediated by binding to VEC rather than to tumor cells, anti-PECAM-1 mAb appears to act independently of tumor type. A modified 3D coculture assay showed that anti-PECAM-1 mAb inhibits the proliferation of PECAM-1-negative tumor cells by altering the concentrations of secreted factors. Our studies indicate that a complex interplay between elements of the TME and advanced tumor metastases directs end-stage metastatic progression. They also suggest that some therapeutic interventions may target late-stage metastases specifically. mAb-based targeting of PECAM-1 represents a TME-targeted therapeutic approach that suppresses the end stages of metastatic progression, until now a refractory clinical entity.
Assuntos
Neoplasias Experimentais/secundário , Molécula-1 de Adesão Celular Endotelial a Plaquetas/fisiologia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/farmacologia , Apoptose , Transplante de Medula Óssea , Caquexia/terapia , Linhagem Celular Tumoral , Proliferação de Células , Progressão da Doença , Células Endoteliais/fisiologia , Feminino , Humanos , Neoplasias Pulmonares/secundário , Neoplasias Pulmonares/terapia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Nus , Camundongos Transgênicos , Neoplasias Experimentais/irrigação sanguínea , Neoplasias Experimentais/patologia , Neoplasias Experimentais/terapia , Neovascularização Patológica , Comunicação Parácrina , Fenótipo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Molécula-1 de Adesão Celular Endotelial a Plaquetas/imunologiaRESUMO
Antiretroviral drugs have dramatically improved the prognosis of HIV-infected patients, with strikingly reduced morbidity and mortality. However, long-term use can be associated with signs of premature aging. Highly active antiretroviral therapy generally comprises two nucleoside reverse transcriptase inhibitors (NRTIs), with one of three additional antiretroviral drug classes, including protease inhibitors (PIs). One commonality between mitochondrial dysfunction (induced by NRTIs) and defects in lamin A (induced by PIs) is they can cause or accelerate cellular senescence, a state of essentially irreversible growth arrest, and the secretion of many bioactive molecules collectively known as the senescence-associated secretory phenotype (SASP). We hypothesized that senescent cells increase following treatment with certain HIV therapies. We compared the effects of two distinct HIV PIs: ritonavir-boosted atazanavir (ATV/r) and ritonavir-boosted darunavir (DRN/r), used in combination treatments for HIV infection. Upon ATV/r, but not DRN/r, treatment, cells arrested growth, displayed multiple features of senescence, and expressed significantly upregulated levels of many SASP factors. Furthermore, mice receiving sustained ATV/r treatment showed an increase in senescent cells and age-related decline in physiological function. However, removing treatment reversed the features of senescence observed in vivo and cell culture. Given how these features disappeared with drug removal, certain features of senescence may not be prognostic as defined by an irreversible growth arrest. Importantly, for patients that are treated or have been treated with ATV/r, our data suggest that switching to another PI that does not promote premature aging conditions (DRN/r) may improve the associated age-related complications.
Assuntos
Senilidade Prematura , Fármacos Anti-HIV , Infecções por HIV , Inibidores da Protease de HIV , Animais , Camundongos , Ritonavir/farmacologia , Ritonavir/uso terapêutico , Inibidores da Protease de HIV/farmacologia , Inibidores da Protease de HIV/uso terapêutico , Infecções por HIV/tratamento farmacológico , Fármacos Anti-HIV/farmacologia , Fármacos Anti-HIV/uso terapêutico , Sulfato de Atazanavir/farmacologia , Sulfato de Atazanavir/uso terapêutico , Darunavir/farmacologia , Darunavir/uso terapêutico , Senescência CelularRESUMO
PURPOSE: This study aimed to investigate the senescent phenotypes of human corneal and conjunctival epithelial cells. METHODS: We examined cell morphology, senescence-associated ß-galactosidase (SA-ß-gal) activity, cell proliferation, and expression of senescence markers (p16 and p21). RNA sequencing analysis was conducted to compare gene expression profiles between senescent and non-senescent cells. Finally, the potential involvement of senescent cells in the pathogenesis of ocular surface diseases was investigated. RESULTS: X-irradiated corneal and conjunctival epithelial cells exhibited typical senescence phenotypes, i.e., flattened morphologies, increased SA-ß-gal activity, decreased cell proliferation, and increased expression of senescence markers, p16 and p21. RNA-seq analysis revealed substantial differences in gene expression profiles between senescent corneal (SCo) and conjunctival epithelial cells (SCj). Moreover, SCj were detected in pathological conjunctival tissues associated with limbal stem cell deficiency (LSCD) due to Stevens-Johnson syndrome or chemical burns, potentially being involved in abnormal differentiation. CONCLUSION: This study highlights the cellular and molecular characteristics of senescent ocular surface cells, particularly in SCj that show abnormal keratin expression, and their potential roles in severe ocular surface diseases and pathology.
Assuntos
Limbo da Córnea , Transcriptoma , Humanos , Limbo da Córnea/patologia , Córnea/metabolismo , Células Epiteliais/metabolismo , Túnica ConjuntivaRESUMO
The longitudinal monitoring of patient circulating tumor DNA (ctDNA) provides a powerful method for tracking the progression, remission, and recurrence of several types of cancer. Often, clinical and research approaches involve the manual review of individual liquid biopsy reports after sampling and genomic testing. Here, we describe a process developed to integrate techniques utilized in data science within a cancer research framework. Using data collection, an analysis that classifies genetic cancer mutations as pathogenic, and a patient matching methodology that identifies the same donor within all liquid biopsy reports, the manual work for research personnel is drastically reduced. Automated dashboards provide longitudinal views of patient data for research studies to investigate tumor progression and treatment efficacy via the identification of ctDNA variant allele frequencies over time.