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Nanoparticles have useful functions due to the characteristics conferred on them by an increase in their specific surface area, and they have already been put into practical use in products in various industrial fields. Although exposure to nanoparticles in daily life is unavoidable for pregnant women, studies that evaluate the toxicity of nanoparticles in pregnant women are lacking. To redress this, we have focused on the placenta and have previously revealed that nanoparticles can show placental toxicity. However, there is still little knowledge regarding the behavior of nanoparticles within placental cells, which would enable us to understand their mode of action. Here, we tried to clarify the intracellular localization of silica nanoparticles in placental cells and how this affects placental toxicity. We analyzed the uptake of silica nanoparticles with a diameter of 10 nm (nSP10) into JEG-3 cells, a human choriocarcinoma cell line. Flow cytometry analysis showed that nSP10 labelled with red fluorescence were taken up into JEG-3 cells, and that pre-treatment with the endocytosis inhibitor cytochalasin D inhibited their uptake, suggesting that nSP10 are taken up into JEG-3 cells by the endocytic pathway. Moreover, confocal microscopy revealed that nSP10 are prominently localized in lysosomes. Staining with LysoTracker showed that nSP10 treatment increased the acidic compartment of JEG-3 cells, suggesting lysosome accumulation and swelling. These results indicate that nSP10 taken into placental cells are transferred to lysosomes and may cause lysosomal dysfunction.
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Microplastics, plastic particles 5â¯mm or less in size, are abundant in the environment; hence, the exposure of humans to microplastics is a great concern. Usually, the surface of microplastics found in the environment has undergone degradation by external factors such as ultraviolet rays and water waves. One of the characteristics of changes caused by surface degradation of microplastics is the introduction of oxygen-containing functional groups. Surface degradation alters the physicochemical properties of plastics, suggesting that the biological effects of environmentally degraded plastics may differ from those of pure plastics. However, the biological effects of plastics introduced with oxygen-containing functional groups through degradation are poorly elucidated owing to the lack of a plastic sample that imitates the degradation state of plastics found in the environment. In this study, we investigated the degradation state of microplastics collected from a beach. Next, we degraded a commercially available polyethylene (PE) particles via vacuum ultraviolet (VUV) irradiation and showed that chemical surface state of PE imitates that of microplastics in the environment. We evaluated the cytotoxic effects of degraded PE samples on immune and epithelial cell lines. We found that VUV irradiation was effective in degrading PE within a short period, and concentration-dependent cytotoxicity was induced by degraded PE in all cell lines. Our results indicate that the cytotoxic effect of PE on different cell types depends on the degree of microplastic degradation, which contributes to our understanding of the effects of PE microplastics on humans.
Assuntos
Microplásticos , Polietileno , Raios Ultravioleta , Poluentes Químicos da Água , Microplásticos/toxicidade , Polietileno/toxicidade , Polietileno/química , Humanos , Poluentes Químicos da Água/toxicidade , Praias , Sobrevivência Celular/efeitos dos fármacos , Animais , Plásticos/toxicidade , Linhagem CelularRESUMO
Various treatment options, such as molecular targeted drugs and immune checkpoint blockades, are available for patients with cancer. However, some cancer types are refractory to molecular targeted therapies or acquire drug resistance after long-term treatment. Thus, ferroptosis, a newly defined type of programmed cell death caused by the iron-dependent accumulation of lipid peroxidation, has gained attention as a novel cancer treatment strategy. Understanding cell-cell interactions in the tumor microenvironment is important for the clinical application of ferroptosis inducers. However, the effects of cell-cell interactions on ferroptosis sensitivity remain unclear. Thus, we aimed to evaluate the effects of macrophage-cancer cell interactions on ferroptosis induction. Coculture experiments showed that conditioned medium prepared from macrophages did not alter the ferroptosis sensitivity of cancer cells. By contrast, coculture via transwell, which enables cell-cell interactions through secretion, increased the sensitivity of cancer cells to ferroptosis inducers. Additionally, direct coculture increased the susceptibility of cancer cells to RSL3-induced ferroptosis. Mechanistically, coculture with macrophages upregulated the levels of intracellular ferrous ions and lipid peroxidation in cancer cells. These findings provide novel insights into the mechanisms by which cell-cell interactions influence ferroptosis induction and application of ferroptosis inducers as a cancer treatment option.
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Microplastics are small pieces of plastic that are less than 5 mm in length. These plastics have been detected in various environments, including the ocean, soil, and air. Their abundance have raised concerns regarding their potential effects on living organisms, including humans. The surface of microplastics degrades due to external factors such as ultraviolet rays and water waves in the environment. Therefore, assessing the biological impact of microplastics and considering their state of degradation is important. Among the physical properties of microplastics, we focused on the chemical degradation of microplastics. Specifically, we used vacuum ultraviolet (VUV) light to accelerate the degradation of polyethylene (PE) and prepared PE samples representing the degradation of PE to varying degrees. The surface properties of PE samples prepared using VUV were similar to those obtained from the environment. Cytotoxicity tests were then used to evaluate the effects of undegraded and degraded PE on cells. We found that the severity of cytotoxicity increased with the extent to which the PE would have been degraded, suggesting that the degree of degradation is strongly linked to the severity of the observed deleterious effects on living organisms. In conclusion, this finding contributes to our understanding of the effects of polyethylene microplastics on the human body.
Assuntos
Microplásticos , Poluentes Químicos da Água , Humanos , Microplásticos/toxicidade , Plásticos , Poluentes Químicos da Água/análise , Monitoramento Ambiental , Polietileno/análise , Propriedades de SuperfícieRESUMO
Microplastics (MPs), defined as plastic particles less than 5 mm in size, are ubiquitous in the environment. The accumulation of MPs in various environmental compartments, such as the ocean, soil, and air, has raised considerable concerns regarding their impact on ecological systems, including marine life and human health. Notably, MPs have been detected in marine organisms such as shellfish and fish, and have even been found in the human body, including in the blood and placenta. Moreover, considering that MPs have been detected in drinking water, human exposure to these particles in daily life is inevitable. To assess the risk posed by MPs to human health, it is essential to consider their physiological and chemical properties, including size, shape, surface modification, and material composition. However, current risk analyses focus primarily on spherical MPs with smooth surfaces, which differ substantially from most of the MPs detected in the environment. Environmental factors, such as ocean waves and ultraviolet radiation, alter the properties of MPs, including size, shape, and surface characteristics. In this review, we summarize current research on MPs, with a particular emphasis on the effects of MP degradation on human health. Furthermore, we generated MPs with surface degradation and evaluated their impact on cell toxicity, along with the underlying biological mechanisms.
Assuntos
Microplásticos , Poluentes Químicos da Água , Animais , Humanos , Microplásticos/toxicidade , Plásticos/toxicidade , Raios Ultravioleta , Poluentes Químicos da Água/toxicidade , Poluentes Químicos da Água/análise , PeixesRESUMO
Placental dysfunction can disrupt pregnancy. However, few studies have assessed the effects of chemical-induced toxicity on placental function. Here, we examined the effects of valproic acid (VPA) as a model chemical on production of hormones and on glucose uptake in human choriocarcinoma cell line BeWo. Cells were treated with forskolin to differentiate into syncytiotrophoblasts, which were then treated with VPA for 72 hr. Real-time RT-PCR analysis showed that VPA significantly increased the mRNA expression of chorionic gonadotropin ß (CGB), a hormone that is produced by the placenta in the first trimester of pregnancy, relative to that in the forskolin-only group. It also suppressed the increase in intracellular glucose uptake and GLUT1 level observed in the forskolin-only group. RNA-seq analysis and pathway database analysis revealed that VPA consistently decreased the level of HIF-1α protein and expression of its downstream target genes HK2 and ADM in the hypoxia pathway. Cobalt chloride, a HIF-1α inducer, inhibited CGB upregulation in VPA-treated cells and rescued VPA-induced suppression of glucose uptake and GLUT1 level. Thus, HIF-1α-mediated elevation of CGB expression and suppression of glucose uptake by VPA is a novel mechanism of placental dysfunction.
Assuntos
Placenta , Ácido Valproico , Gravidez , Feminino , Humanos , Ácido Valproico/toxicidade , Placenta/metabolismo , Colforsina/metabolismo , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 1/metabolismo , Glucose/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Linhagem Celular TumoralRESUMO
EGFR-specific tyrosine kinase inhibitors (TKIs), especially osimertinib, have changed lung cancer therapy, but secondary mutations confer drug resistance. Because other EGFR mutations promote dimerization-independent active conformations but L858R strictly depends on receptor dimerization, we herein evaluate the therapeutic potential of dimerization-inhibitory monoclonal antibodies (mAbs), including cetuximab. This mAb reduces viability of cells expressing L858R-EGFR and blocks the FOXM1-aurora survival pathway, but other mutants show no responses. Unlike TKI-treated patient-derived xenografts, which relapse post osimertinib treatment, cetuximab completely prevents relapses of L858R+ tumors. We report that osimertinib's inferiority associates with induction of mutagenic reactive oxygen species, whereas cetuximab's superiority is due to downregulation of adaptive survival pathways (e.g., HER2) and avoidance of mutation-prone mechanisms that engage AXL, RAD18, and the proliferating cell nuclear antigen. These results identify L858R as a predictive biomarker, which may pave the way for relapse-free mAb monotherapy relevant to a large fraction of patients with lung cancer.
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Receptores ErbB , Neoplasias Pulmonares , Humanos , Cetuximab/farmacologia , Cetuximab/uso terapêutico , Receptores ErbB/genética , Inibidores de Proteínas Quinases/farmacologia , Recidiva Local de Neoplasia/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Anticorpos Monoclonais/uso terapêutico , Biomarcadores , Proteínas de Ligação a DNA , Ubiquitina-Proteína LigasesRESUMO
Developing a generalized model for a robust prediction of nanotoxicity is critical for designing safe nanoparticles. However, complex toxicity mechanisms of nanoparticles in biological environments, such as biomolecular corona formation, prevent a reliable nanotoxicity prediction. This is exacerbated by the potential evaluation bias caused by internal validation, which is not fully appreciated. Herein, we propose an evidence-based prediction method for distinguishing between cytotoxic and noncytotoxic nanoparticles at a given condition by uniting literature data mining and machine learning. We illustrate the proposed method for amorphous silica nanoparticles (SiO2-NPs). SiO2-NPs are currently considered a safety concern; however, they are still widely produced and used in various consumer products. We generated the most diverse attributes of SiO2-NP cellular toxicity to date, using >100 publications, and built predictive models, with algorithms ranging from linear to nonlinear (deep neural network, kernel, and tree-based) classifiers. These models were validated using internal (4124-sample) and external (905-sample) data sets. The resultant categorical boosting (CatBoost) model outperformed other algorithms. We then identified 13 key attributes, including concentration, serum, cell, size, time, surface, and assay type, which can explain SiO2-NP toxicity, using the Shapley Additive exPlanation values in the CatBoost model. The serum attribute underscores the importance of nanoparticle-corona complexes for nanotoxicity prediction. We further show that internal validation does not guarantee generalizability. In general, safe SiO2-NPs can be obtained by modifying their surfaces and using low concentrations. Our work provides a strategy for predicting and explaining the toxicity of any type of engineered nanoparticles in real-world practice.
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Nanopartículas , Dióxido de Silício , Dióxido de Silício/toxicidade , Nanopartículas/toxicidade , Algoritmos , Redes Neurais de ComputaçãoRESUMO
Among triple-negative breast cancer (TNBC) subtypes, the basal-like 2 (BL2) subtype shows the lowest survival rate and the highest risk of metastasis after treatment with chemotherapy. Research has shown that αB-crystallin (CRYAB) is more highly expressed in the basal-like subtypes than in the other subtypes and is associated with brain metastasis in TNBC patients. We therefore hypothesized that αB-crystallin is associated with increased cell motility in the BL2 subtype after treatment with chemotherapy. Here, we evaluated the effect of fluorouracil (5-FU), a typical chemotherapy for the treatment of TNBC, on cell motility by utilizing a cell line with high αB-crystallin expression (HCC1806). A wound healing assay revealed that 5-FU significantly increased cell motility in HCC1806 cells, but not in MDA-MB-231 cells, which have low αB-crystallin expression. Also, cell motility was not increased by 5-FU treatment in HCC1806 cells harboring stealth siRNA targeting CRYAB. In addition, the cell motility of MDA-MB-231 cells overexpressing αB-crystallin was significantly higher than that of MDA-MB-231 cells harboring a control vector. Thus, 5-FU increased cell motility in cell lines with high, but not low, αB-crystallin expression. These results suggest that 5-FU-induced cell migration is mediated by αB-crystallin in the BL2 subtype of TNBC.
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Neoplasias de Mama Triplo Negativas , Humanos , Neoplasias de Mama Triplo Negativas/metabolismo , Cadeia B de alfa-Cristalina/genética , Fluoruracila , Movimento Celular , Linhagem Celular TumoralRESUMO
Tyrosine kinase inhibitors of epidermal growth factor receptor (EGFR-TKIs), such as osimertinib, show great success in non-small-cell lung cancer patients with EGFR mutated tumors. However, almost all patients develop resistance to EGFR-TKIs owing to secondary EGFR mutations. Although genetic and irreversible resistance mechanisms have been proposed, little is known about non-genetic and reversible resistance mechanisms. From this perspective, a recent study revealed that acute drug exposure generates drug-tolerant persister cells (DTPs) as a form of non-genetic resistance. However, the biological characteristics of DTPs remain unclear. As lipid peroxidation is related to cancer progression and drug resistance, we focused on ferroptosis, namely programmed cell death induced by the accumulation of lipid peroxides, in DTPs. We examined the biological characteristics of ferroptosis in osimertinib-mediated DTPs derived from PC9 lung adenocarcinoma cells. Unlike PC9 cells, established PC9 DTPs were highly sensitive to the ferroptosis inducer RSL3. Accordingly, PC9 DTPs had increased levels of lipid reactive oxygen species and ferrous ion accumulation. Moreover, RSL3-mediated cell death in PC9 DTPs was completely rescued by treatment with the iron chelator deferoxamine. These results suggest that PC9 DTPs showed increased intracellular ferrous ion accumulation and were susceptible to ferroptosis.
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Carcinoma Pulmonar de Células não Pequenas , Ferroptose , Neoplasias Pulmonares , Humanos , Neoplasias Pulmonares/patologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Resistencia a Medicamentos Antineoplásicos , Inibidores de Proteínas Quinases/farmacologia , Linhagem Celular Tumoral , Receptores ErbB/genética , MutaçãoRESUMO
Although molecular targeted drugs are significantly effective in many types of cancer treatment, almost all patients suffer from drug resistance. For instance, non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutation invariably develop resistance to EGFR tyrosine kinase inhibitors (EGFR-TKIs) and melanoma patients with BRAF mutation develop resistance to BRAF inhibitors. Mechanistically, genetic and irreversible resistance mechanisms have been studied for more than a decade, while non-mutational and reversible resistance mechanisms are yet to be clearly understood. Since drug tolerant persisters (DTPs), which emerge at the beginning of the drug treatment, have been reported in 2010, several non-mutational tolerance mechanisms have been reported by various researchers. Furthermore, with the advancement in single cell sequencing technology, increasing attention has been drawn towards the investigation of the heterogeneous characteristics of drug tolerant cell populations. Here, we describe the recent advances in non-mutational drug tolerant mechanisms toward the molecular targeted drugs. In our study, we tried to elucidate the unconventional resistance mechanisms by utilizing newly approved EGFR-TKI, dacomitinib. Our established drug resistant cells did not gain new mutation in EGFR even after long time exposure to the drug. In addition, the drug resistance vanished when resistant cells were implanted in mice, which indicates that mechanisms conferring drug sensitivity might be host-dependent. Thus, our study may provide a new insight into non-mutational drug tolerant mechanisms.
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Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Animais , Camundongos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Proteínas Proto-Oncogênicas B-raf , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Receptores ErbB/genética , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Resistência a MedicamentosRESUMO
Bioconjugation with polyethylene glycol (PEG) is important for protein drug development as it has improved biological stability. In contrast, proteins including PEGylated ones are susceptible to physicochemical stresses. Particularly, protein drugs in solution may form aggregates or subvisible particles if they are exposed to dropping stress during transportation. However, many PEGylation studies have focused on its usefulness, such as the extension of half-life in blood, and changes in the physical properties or biological responses of PEGylated proteins under dropping stress remain unexplored. Here, we prepared four PEGylated ovalbumin (PEG-OVA) molecules conjugated with different lengths (5 or 20 kDa) and numbers (large [L] or small [S]) of PEG, analyzed the formation of subvisible particles under dropping stress, and examined their impact on antibody production and clearance. Under dropping stress, the aggregated particle concentration of 20 kDa PEG-OVA (S) and (L) solutions was approximately 3-fold that of the OVA solution. Moreover, administration of 20 kDa PEG-OVA with dropping stress induced anti-PEG antibody production and clearance of PEG-OVA. As a mechanism, dropping stress could enhance the uptake of 20 kDa PEG-OVA (L) by macrophages. These findings could provide insights into proper transportation conditions to ensure the quality of PEGylated protein drugs.
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Formação de Anticorpos , Polietilenoglicóis , Animais , Camundongos , Ovalbumina , Preparações Farmacêuticas , Polietilenoglicóis/química , ProteínasRESUMO
The specific human epidermal growth factor receptor 2 (HER2)-targeting monoclonal antibody trastuzumab shows considerable clinical efficacy in patients with HER2-overexpressing breast cancer. However, about 20% of patients who receive trastuzumab in the adjuvant setting relapse, and approximately half of patients with metastatic HER2-positive breast cancer develop resistance to trastuzumab within 1 year. Although the mechanism of trastuzumab resistance has been explored broadly, whether and how angiogenesis participates in trastuzumab resistance is unclear. Here, we examined the association between angiogenesis and trastuzumab resistance by using a trastuzumab-resistant cell line (SKBR3-TR). Compared with that from the parental trastuzumab-sensitive SKBR3 cells, the culture supernatant from SKBR3-TR cells significantly increased the sprouting of endothelial cells. To identify intercellular features that contribute to the induction of endothelial tube formation, proteomics revealed that α-crystallin B chain (αB-crystallin) was upregulated in SKBR3-TR cells. Moreover, silencing of αB-crystallin significantly repressed SKBR3-TR-induced tube formation, and knockdown of αB-crystallin in SKBR3-TR cells suppressed the activation of mechanistic target of rapamycin (mTOR) in endothelial cells. In addition, treatment with rapamycin, an inhibitor of mTOR, reversed the SKBR3-TR-induced promotion of tube formation. In summary, αB-crystallin enhanced the ability of SKBR3-TR cells to activate mTOR in endothelial cells and thus promote angiogenesis.
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Neoplasias da Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Células Endoteliais/metabolismo , Neovascularização Fisiológica , Serina-Treonina Quinases TOR/metabolismo , Trastuzumab/uso terapêutico , Cadeia B de alfa-Cristalina/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Meios de Cultivo Condicionados/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Feminino , Humanos , Neovascularização Fisiológica/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Trastuzumab/farmacologiaRESUMO
Opportunities for the exposure of pregnant women to engineered nanoparticles have been increasing with the expanding use of these materials. Therefore, there are concerns that nanoparticles could have adverse effects on the establishment and maintenance of pregnancy. The effects of nanoparticles on the mother and fetus have been evaluated from this perspective, but there is still little knowledge about the effects on placentation and function acquisition, which are essential for the successful establishment and maintenance of pregnancy. Formation of the syncytiotrophoblast is indispensable for the acquisition of placental function, and impairment of syncytialization inevitably affects pregnancy outcomes. Here, we assessed the effect of nanoparticles on placental formation by using forskolin-treated BeWo cells, a typical in vitro model of trophoblast syncytialization. Immunofluorescence staining analysis revealed that silver nanoparticles with a diameter of 10 nm (nAg10) (at 0.156 µg/mL) significantly decreased the proportion of syncytialized BeWo cells, but gold nanoparticles with a diameter of 10 nm did not. Consistently, only nAg10 (at 0.156 µg/mL) significantly suppressed forskolin-induced elevation of CGB and SDC1 mRNA expression levels and human chorionic gonadotropin ß production in a dose-dependent manner; these molecules are all markers of syncytialization. Besides, nAg10 significantly decreased the expression of ERVFRD-1, which encodes proteins associated with cell fusion. Moreover, nAg10 tended to suppress the expression of sFlt-1 e15a, a placental angiogenesis marker. Collectively, our data suggest that nAg10 could suppress formation of the syncytiotrophoblast and that induce placental dysfunction and the following poor pregnancy outcomes.
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Nanopartículas Metálicas , Placenta , Feminino , Gravidez , Humanos , Colforsina/farmacologia , Prata , OuroRESUMO
Unlike early transcriptional responses to mitogens, later events are less well-characterized. Here, we identified delayed down-regulated genes (DDGs) in mammary cells after prolonged treatment with epidermal growth factor (EGF). The expression of these DDGs was low in mammary tumors and correlated with prognosis. The proteins encoded by several DDGs directly bind to and inactivate oncoproteins and might therefore act as tumor suppressors. The transcription factor teashirt zinc finger homeobox 2 (TSHZ2) is encoded by a DDG, and we found that overexpression of TSHZ2 inhibited tumor growth and metastasis and accelerated mammary gland development in mice. Although the gene TSHZ2 localizes to a locus (20q13.2) that is frequently amplified in breast cancer, we found that hypermethylation of its promoter correlated with down-regulation of TSHZ2 expression in patients. Yeast two-hybrid screens and protein-fragment complementation assays in mammalian cells indicated that TSHZ2 nucleated a multiprotein complex containing PRC1/Ase1, cyclin B1, and additional proteins that regulate cytokinesis. TSHZ2 increased the inhibitory phosphorylation of PRC1, a key driver of mitosis, mediated by cyclin-dependent kinases. Furthermore, similar to the tumor suppressive transcription factor p53, TSHZ2 inhibited transcription from the PRC1 promoter. By recognizing DDGs as a distinct group in the transcriptional response to EGF, our findings uncover a group of tumor suppressors and reveal a role for TSHZ2 in cell cycle regulation.
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Neoplasias da Mama , Proteínas de Ciclo Celular , Citocinese , Proteínas de Homeodomínio/genética , Animais , Mama , Neoplasias da Mama/genética , Fator de Crescimento Epidérmico/genética , Feminino , Genes Supressores de Tumor , Humanos , CamundongosRESUMO
Lung cancers driven by mutant forms of EGFR invariably develop resistance to kinase inhibitors, often due to secondary mutations. Here we describe an unconventional mechanism of resistance to dacomitinib, a newly approved covalent EGFR kinase inhibitor, and uncover a previously unknown step of resistance acquisition. Dacomitinib-resistant (DR) derivatives of lung cancer cells were established by means of gradually increasing dacomitinib concentrations. These DR cells acquired no secondary mutations in the kinase or other domains of EGFR. Along with resistance to other EGFR inhibitors, DR cells acquired features characteristic to epithelial-mesenchymal transition, including an expanded population of aldehyde dehydrogenase-positive cells and upregulation of AXL, a receptor previously implicated in drug resistance. Unexpectedly, when implanted in animals, DR cells reverted to a dacomitinib-sensitive state. Nevertheless, cell lines derived from regressing tumors displayed renewed resistance when cultured in vitro. Three-dimensional and cocultures along with additional analyses indicated lack of involvement of hypoxia, fibroblasts, and immune cells in phenotype reversal, implying that other host-dependent mechanisms might nullify nonmutational modes of resistance. Thus, similar to the phenotypic resistance of bacteria treated with antibiotics, the reversible resisters described here likely evolve from drug-tolerant persisters and give rise to the irreversible, secondary mutation-driven nonreversible resister state. SIGNIFICANCE: This study reports that stepwise acquisition of kinase inhibitor resistance in lung cancers driven by mutant EGFR comprises a nonmutational, reversible resister state. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/14/3862/F1.large.jpg.
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Inibidores de Proteínas Quinases/uso terapêutico , Animais , Feminino , Humanos , Camundongos , Camundongos Nus , Fenótipo , Inibidores de Proteínas Quinases/farmacologiaRESUMO
Currently, the only therapeutic choice for the treatment of triple-negative breast cancer (TNBC) is chemotherapy. In TNBC, despite strong preclinical data, clinical trials of molecular targeted drugs, such as the Src tyrosine kinase inhibitor dasatinib, have failed because of the heterogeneity of TNBC cells. Here, we examined the mechanism of intrinsic resistance to dasatinib in five TNBC cell lines. First, we divided the TNBC cell lines into those sensitive or resistant to dasatinib and found that activation of Src was inhibited in all of the cell lines. In contrast, we found that dasatinib inhibited Akt phosphorylation in only the dasatinib-sensitive cell lines. Consequently, we found that combination treatment with dasatinib and an inhibitor of Akt or mTOR suppressed cell proliferation more than did either monotherapy in the dasatinib-resistant cell lines. Finally, to mimic intrinsic resistance, we established a dasatinib-tolerant TNBC cell line. In this cell line, the combinational effect of Akt/mTOR inhibition with dasatinib was observed, as it was in the cell lines with intrinsic resistance. Together, the present results show that the effect of dasatinib in TNBC is independent of Src inhibition, and that Akt/mTOR inhibition might be an effective strategy to overcome TNBC cells with intrinsic dasatinib resistance.
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Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Dasatinibe/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Serina-Treonina Quinases TOR/antagonistas & inibidores , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Sirolimo/farmacologia , Neoplasias de Mama Triplo Negativas/genéticaRESUMO
In non-small-cell lung cancer, mutation of epidermal growth factor receptor (EGFR) stimulates cell proliferation and survival. EGFR tyrosine kinase inhibitors (EGFR-TKIs) such as erlotinib are used as first-line therapy with drastic and immediate effectiveness. However, the disease eventually progresses in most cases within a few years due to the development of drug resistance. Here, we explored the role of progesterone membrane component 1 (PGRMC1) in acquired resistance to erlotinib and addressed the molecular mechanism of EGFR-TKI resistance induced by PGRMC1. The erlotinib-sensitive cell line PC9 (derived from non-small-cell lung cancer) and the erlotinib-resistant cell line PC9/ER were used. In proteomic and immunoblotting analyses, the PGRMC1 level was higher in PC9/ER cells than in PC9 cells. WST-8 assay revealed that inhibition of PGRMC1 by siRNA or AG-205, which alters the spectroscopic properties of the PGRMC1-heme complex, in PC9/ER cells increased the sensitivity to erlotinib, and overexpression of PGRMC1 in PC9 cells reduced their susceptibility to erlotinib. In the presence of erlotinib, immunoprecipitation assay showed that AG-205 suppressed the interaction between EGFR and PGRMC1 in PC9/ER cells. AG-205 decreased the expression of ß-catenin, accompanied by up-regulation of IκBα (also known as NFKBIA). Furthermore, AG-205 reduced the expression of ß-TrCP (also known as BTRC), suggesting that PGRMC1 enhanced the crosstalk between NF-κB (also known as NFKB) signaling and Wnt/ß-catenin signaling in an erlotinib-dependent manner. Finally, treatment with the Wnt/ß-catenin inhibitor XAV939 enhanced the sensitivity of PC9/ER cells to erlotinib. These results suggest that PGRMC1 conferred resistance to erlotinib through binding with EGFR in PC9/ER cells, initiating crosstalk between the Wnt/ß-catenin and NF-κB pathways.
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Adenocarcinoma/genética , Adenocarcinoma/patologia , Antineoplásicos , Resistencia a Medicamentos Antineoplásicos/genética , Cloridrato de Erlotinib/farmacologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Proteínas de Membrana/metabolismo , NF-kappa B/metabolismo , Inibidores de Proteínas Quinases , Receptores de Progesterona/metabolismo , Transdução de Sinais/genética , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/genética , beta Catenina/metabolismo , Adenocarcinoma/metabolismo , Linhagem Celular Tumoral , Receptores ErbB/metabolismo , Humanos , Neoplasias Pulmonares/metabolismoRESUMO
The glucocorticoid receptor (GR) acts as a ubiquitous cortisol-dependent transcription factor (TF). To identify co-factors, we used protein-fragment complementation assays and found that GR recognizes FLI1 and additional ETS family proteins, TFs relaying proliferation and/or migration signals. Following steroid-dependent translocation of FLI1 and GR to the nucleus, the FLI1-specific domain (FLS) binds with GR and strongly enhances GR's transcriptional activity. This interaction has functional consequences in Ewing sarcoma (ES), childhood and adolescence bone malignancies driven by fusions between EWSR1 and FLI1. In vitro, GR knockdown inhibited the migration and proliferation of ES cells, and in animal models, antagonizing GR (or lowering cortisol) retarded both tumor growth and metastasis from bone to lung. Taken together, our findings offer mechanistic rationale for repurposing GR-targeting drugs for the treatment of patients with ES.