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The mechanistic target of rapamycin complex 1 (mTORC1) integrates inputs from growth factors and nutrients, but how mTORC1 autoregulates its activity remains unclear. The MiT/TFE transcription factors are phosphorylated and inactivated by mTORC1 following lysosomal recruitment by RagC/D GTPases in response to amino acid stimulation. We find that starvation-induced lysosomal localization of the RagC/D GAP complex, FLCN:FNIP2, is markedly impaired in a mTORC1-sensitive manner in renal cells with TSC2 loss, resulting in unexpected TFEB hypophosphorylation and activation upon feeding. TFEB phosphorylation in TSC2-null renal cells is partially restored by destabilization of the lysosomal folliculin complex (LFC) induced by FLCN mutants and is fully rescued by forced lysosomal localization of the FLCN:FNIP2 dimer. Our data indicate that a negative feedback loop constrains amino acid-induced, FLCN:FNIP2-mediated RagC activity in renal cells with constitutive mTORC1 signaling, and the resulting MiT/TFE hyperactivation may drive oncogenesis with loss of the TSC2 tumor suppressor.
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Aminoácidos , Lisosomas , Aminoácidos/metabolismo , Retroalimentación , Lisosomas/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismoRESUMEN
GPNMB (glycoprotein nonmetastatic B) and other TFE3/TFEB transcriptional targets have been proposed as markers for microphthalmia (MiT) translocation renal cell carcinomas (tRCCs). We recently demonstrated that constitutive mTORC1 activation via TSC1/2 loss leads to increased activity of TFE3/TFEB, suggesting that the pathogenesis and molecular markers for tRCCs and TSC1/2-associated tumors may be overlapping. We examined GPNMB expression in human kidney and angiomyolipoma (AML) cell lines with TSC2 and/or TFE3/TFEB loss produced using CRISPR-Cas9 genome editing as well as in a mouse model of Tsc2 inactivation-driven renal tumorigenesis. Using an automated immunohistochemistry (IHC) assay for GPNMB, digital image analysis was employed to quantitatively score expression in clear cell RCC (ccRCC, n = 87), papillary RCC (papRCC, n = 53), chromophobe RCC (chRCC, n = 34), oncocytoma (n = 4), TFE3- or TFEB-driven tRCC (n = 56), eosinophilic solid and cystic RCC (ESC, n = 6), eosinophilic vacuolated tumor (EVT, n = 4), and low-grade oncocytic tumor (LOT, n = 3), as well as AML (n = 29) and perivascular epithelioid cell tumors (PEComas, n = 8). In cell lines, GPNMB was upregulated following TSC2 loss in a MiT/TFE- and mTORC1-dependent fashion. Renal tumors in Tsc2+/- A/J mice showed upregulation of GPNMB compared with normal kidney. Mean GPNMB expression was significantly higher in tRCC than in ccRCC (p < 0.0001), papRCC (p < 0.0001), and chRCC (p < 0.0001). GPNMB expression in TSC1/2/MTOR alteration-associated renal tumors (including ESC, LOT, AML, and PEComa) was comparable to that in tRCC. The immunophenotype of tRCC and TSC1/2/MTOR alteration-associated renal tumors is highly overlapping, likely due to the increased activity of TFE3/TFEB in both, revealing an important caveat regarding the use of TFE3/TFEB-transcriptional targets as diagnostic markers. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
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Carcinoma de Células Renales , Neoplasias Renales , Leucemia Mieloide Aguda , Microftalmía , Neoplasias de Células Epitelioides Perivasculares , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/genética , Carcinoma de Células Renales/patología , Proteínas del Ojo , Femenino , Humanos , Neoplasias Renales/patología , Leucemia Mieloide Aguda/genética , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina , Glicoproteínas de Membrana/genética , Ratones , Microftalmía/genética , Neoplasias de Células Epitelioides Perivasculares/genética , Serina-Treonina Quinasas TOR/genética , Factores de Transcripción/genética , Translocación Genética , Esclerosis TuberosaRESUMEN
Biguanide drugs (metformin and phenformin) have drawn interest for potential cancer treatments, and laboratory studies show that some cancer cells are selectively sensitive to growth-inhibitory effects of biguanides. Examining metabolic pathways affected by biguanide treatments in cancer cells that are highly sensitive to biguanides, we found that biguanide treatment depletes cellular levels of both aspartate and NAD+. Experiments to replenish these metabolites or block steps of the aspartate-malate shuttle suggest that depletion of both metabolites, rather than either aspartate of NAD+ individually, is critical for growth-inhibitory effects of biguanide exposure. Even in sensitive cancer cells, though, biguanide treatment alone over a broad range of doses only inhibits cell replication without significantly affecting cell viability. Noting that clinical observations of biguanide efficacy have used combinations of agents that typically include cisplatin, we found that biguanide treatment at a cytostatic level substantially decreases survival of lung cancer and breast cancer cells when co-treated with cisplatin at doses that alone are also non-cytotoxic. This striking enhancement of cisplatin toxicity by biguanides depends on reductions of levels of NAD+ and aspartate, since addition of either of these metabolites prevented this potentiation of cisplatin cytotoxicity. Thus, biguanide drugs can have cytotoxic effects when used in combination with other cancer drugs, such as cisplatin, and depleting cellular levels of NAD+ and aspartate is critical for enhancing the cytotoxicity of cisplatin by biguanide drugs in sensitive cancer cells.
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Antineoplásicos , Metformina , Neoplasias , Preparaciones Farmacéuticas , Ácido Aspártico , Cisplatino , NADRESUMEN
Neuroendocrine prostate cancer (NEPC) is a rare but aggressive histologic variant of prostate cancer that responds poorly to androgen deprivation therapy. Hybrid NEPC-adenocarcinoma (AdCa) tumors are common, often eluding accurate pathologic diagnosis and requiring ancillary markers for classification. We recently performed an outlier-based meta-analysis across a number of independent gene expression microarray datasets to identify novel markers that differentiate NEPC from AdCa, including up-regulation of insulinoma-associated protein 1 (INSM1) and loss of Yes-associated protein 1 (YAP1). Here, using diverse cancer gene expression datasets, we show that Hippo pathway-related genes, including YAP1, are among the top down-regulated gene sets with expression of the neuroendocrine transcription factors, including INSM1. In prostate cancer cell lines, transgenic mouse models, and human prostate tumor cohorts, we confirm that YAP1 RNA and YAP1 protein expression are silenced in NEPC and demonstrate that the inverse correlation of INSM1 and YAP1 expression helps to distinguish AdCa from NEPC. Mechanistically, we find that YAP1 loss in NEPC may help to maintain INSM1 expression in prostate cancer cell lines and we further demonstrate that YAP1 silencing likely occurs epigenetically, via CpG hypermethylation near its transcriptional start site. Taken together, these data nominate two additional markers to distinguish NEPC from AdCa and add to data from other tumor types suggesting that Hippo signaling is tightly reciprocally regulated with neuroendocrine transcription factor expression. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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Biomarcadores de Tumor/metabolismo , Carcinoma Neuroendocrino/patología , Neoplasias de la Próstata Resistentes a la Castración/patología , Proteínas Represoras/metabolismo , Proteínas Señalizadoras YAP/metabolismo , Animales , Biomarcadores de Tumor/análisis , Carcinoma Neuroendocrino/metabolismo , Xenoinjertos , Humanos , Masculino , Ratones , Neoplasias de la Próstata Resistentes a la Castración/metabolismoRESUMEN
Cancer cells hijack autophagy pathway to evade anti-cancer therapeutics. Many molecular signaling pathways associated with drug-resistance converge on autophagy induction. Honokiol (HNK), a natural phenolic compound purified from Magnolia grandiflora, has recently been shown to impede breast tumorigenesis and, in the present study, we investigated whether breast cancer cells evoke autophagy to modulate therapeutic efficacy and functional networks of HNK. Indeed, breast cancer cells exhibit increased autophagosomes-accumulation, MAP1LC3B-II/LC3B-II-conversion, expression of ATG proteins as well as elevated fusion of autophagosomes and lysosomes upon HNK treatment. Breast cancer cells treated with HNK demonstrate significant growth inhibition and apoptotic induction, and these biological processes are blunted by macroautophagy/autophagy. Consequently, inhibiting autophagosome formation, abrogating autophagosome-lysosome fusion or genetic-knockout of BECN1 and ATG7 effectively increase HNK-mediated apoptotic induction and growth inhibition. Next, we explored the functional impact of tumor suppressor STK11 in autophagy induction in HNK-treated cells. STK11-silencing abrogates LC3B-II-conversion, and blocks autophagosome/lysosome fusion and lysosomal activity as illustrated by LC3B-Rab7 co-staining and DQ-BSA assay. Our results exemplify the cytoprotective nature of autophagy invoked in HNK-treated breast cancer cells and put forth the notion that a combined strategy of autophagy inhibition with HNK would be more effective. Indeed, HNK and chloroquine (CQ) show synergistic inhibition of breast cancer cells and HNK-CQ combination treatment effectively inhibits breast tumorigenesis and metastatic progression. Tumor-dissociated cells from HNK-CQ treated tumors exhibit abrogated invasion and migration potential. Together, these results implicate that breast cancer cells undergo cytoprotective autophagy to circumvent HNK and a combined treatment with HNK and CQ can be a promising therapeutic strategy for breast cancer.
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Disruption of the KEAP1-NRF2 pathway results in the transactivation of NRF2 target genes, consequently inducing cell proliferation and other phenotypic changes in cancer cells. Here, we demonstrated that GULP1 was a KEAP1-binding protein that maintained actin cytoskeleton architecture and helped KEAP1 to sequester NRF2 in the cytoplasm. In urothelial carcinoma of the bladder (UCB), silencing of GULP1 facilitated the nuclear accumulation of NRF2, led to constitutive activation of NRF2 signaling, and conferred resistance to the platinum drug cisplatin. Knockdown of GULP1 in UCB cells promoted tumor cell proliferation in vitro and enhanced tumor growth in vivo. In primary UCB, GULP1 silencing was more prevalent in muscle-invasive UCB compared to nonmuscle-invasive UCB. GULP1 knockdown cells showed resistance to cisplatin treatment. In parallel with decreased GULP1 expression, we observed increased expression of NRF2, HMOX1, and other candidate antioxidant genes in cisplatin-resistant cells. Furthermore, low or no expression of GULP1 was observed in most cisplatin nonresponder cases. Silencing of GULP1 was associated with GULP1 promoter hypermethylation in cell lines and primary tumors, and a high frequency of GULP1 promoter methylation was observed in multiple sets of primary clinical UCB samples. Together, our findings demonstrate that GULP1 is a KEAP1-binding protein that regulates KEAP1-NRF2 signaling in UCB and that promoter hypermethylation of GULP1 is a potential mechanism of GULP1 silencing.
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Proteínas Adaptadoras Transductoras de Señales/genética , Carcinoma de Células Transicionales/genética , Regulación Neoplásica de la Expresión Génica , Proteína 1 Asociada A ECH Tipo Kelch/genética , Factor 2 Relacionado con NF-E2/genética , Transducción de Señal/genética , Neoplasias de la Vejiga Urinaria/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Carcinoma de Células Transicionales/metabolismo , Carcinoma de Células Transicionales/patología , Línea Celular Tumoral , Proliferación Celular/genética , Metilación de ADN , Células HEK293 , Humanos , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Factor 2 Relacionado con NF-E2/metabolismo , Trasplante Heterólogo , Carga Tumoral/genética , Neoplasias de la Vejiga Urinaria/metabolismo , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
OBJECTIVE: We investigated the effects of chronic waterpipe (WP) smoke on pulmonary function and immune response in a murine model using a research-grade WP and the effects of acute exposure on the regulation of immediate-early genes (IEGs). METHODS: WP smoke was generated using three WP smoke puffing regimens based on the Beirut regimen. WP smoke samples generated under these puffing regimens were quantified for nicotine concentration. Mice were chronically exposed for 6 months followed by assessment of pulmonary function and airway inflammation. Transcriptomic analysis using RNAseq was conducted after acute exposure to characterise the IEG response. These biomarkers were then compared with those generated after exposure to dry smoke (without water added to the WP bowl). RESULTS: We determined that nicotine composition in WP smoke ranged from 0.4 to 2.5 mg per puffing session. The lung immune response was sensitive to the incremental severity of chronic exposure, with modest decreases in airway inflammatory cells and chemokine levels compared with air-exposed controls. Pulmonary function was unmodified by chronic WP exposure. Acute WP exposure was found to activate the immune response and identified known and novel IEG as potential biomarkers of WP exposure. CONCLUSION: Chronic exposure to WP smoke leads to immune suppression without significant changes to pulmonary function. Transcriptomic analysis of the lung after acute exposure to WP smoke showed activation of the immune response and revealed IEGs that are common to WP and dry smoke, as well as pools of IEGs unique to each exposure, identifying potential biomarkers specific to WP exposure.
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Genes Inmediatos-Precoces , Pulmón/inmunología , Nicotina/análisis , Fumar en Pipa de Agua/inmunología , Animales , Biomarcadores/metabolismo , Femenino , Ratones , Ratones Endogámicos C57BL , Pipas de AguaRESUMEN
Withaferin A (WFA), a steroidal lactone, negatively regulates breast cancer growth however, its mechanisms of action remain largely elusive. We found that WFA blocks autophagy flux and lysosomal proteolytic activity in breast cancer cells. WFA increases accumulation of autophagosomes, LC3B-II conversion, expression of autophagy-related proteins and autophagosome/lysosome fusion. Autolysosomes display the characteristics of acidic compartments in WFA-treated cells; however, the protein degradation activity of lysosomes is inhibited. Blockade of autophagic flux reduces the recycling of cellular fuels leading to insufficient substrates for tricarboxylic acid (TCA) cycle and impaired oxidative phosphorylation. WFA decreases expression and phosphorylation of lactate dehydrogenase, the key enzyme that catalyzes pyruvate-to-lactate conversion, reduces adenosine triphosphate levels and increases AMP-activated protein kinase (AMPK) activation. AMPK inhibition abrogates while AMPK activation potentiates WFA's effect. WFA and 2-deoxy-d-glucose combination elicits synergistic inhibition of breast cancer cells. Genetic knockout of BECN1 and ATG7 fails to rescue cells from WFA treatment; in contrast, addition of methyl pyruvate to supplement TCA cycle protects WFA-treated cells. Together, these results implicate that WFA is a potent lysosomal inhibitor; energetic impairment is required for WFA-induced apoptosis and growth inhibition and combining WFA and 2-DG is a promising therapeutic strategy for breast cancer.
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BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is common in East Asia and also is often deadly. We sought to determine whether measuring the discoidin domain receptor-1 (DDR1)-both total and phosphorylated proteins-could improve our ability to predict recurrence in ESCC. MATERIALS AND METHODS: Total DDR1 and phosphorylated DDR1 (pDDR1) were measured using semiquantitative immunohistochemistry in a cohort of 60 patients with ESCC. Association between these immunohistochemical measurements and standard clinical-pathological variables such as patient recurrence-free survival was examined using univariate and multivariate analyses. RESULTS: Six patients (10.0%) had regional recurrence and eight patients (13.3%) had distant recurrence. In univariate analysis, early disease recurrence correlated with intense staining of total DDR1 (P = 0.03) as well as intense staining of pDDR1 (P < 0.001). On multivariate analysis, only regional lymph node metastasis (P = 0.04, HR = 4.20) and intensity of pDDR1 immunohistochemistry (P = 0.03, HR = 4.27) emerged as significant independent prognostic factors for recurrence. CONCLUSIONS: This study suggests that immunohistochemical measurements of both the DDR1 protein and pDDR1 can provide prognostic value in ESCC, even when other clinical and pathological factors are also being considered.
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Carcinoma de Células Escamosas/metabolismo , Receptor con Dominio Discoidina 1/metabolismo , Neoplasias Esofágicas/metabolismo , Anciano , Anciano de 80 o más Años , Biomarcadores/metabolismo , Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/mortalidad , Neoplasias Esofágicas/diagnóstico , Neoplasias Esofágicas/mortalidad , Femenino , Humanos , Japón/epidemiología , Masculino , Persona de Mediana Edad , Fosforilación , Pronóstico , Estudios RetrospectivosRESUMEN
Autophagy can serve as a mechanism for survival of cells during nutrient deprivation by recycling cellular macromolecules and organelles transiently to provide essential metabolic substrates. However, autophagy itself causes metabolic stress to cells, and other cellular protective mechanisms likely cooperate with autophagy to promote cell survival during nutrient deprivation. In this study, we explored protective mechanisms in breast cancer cells in the setting of glucose deprivation. While breast cancer cells (MCF7 and T47D) survive in glucose-free medium for three days or more, autophagy is induced in this setting. Blocking autophagy pharmacologically with chloroquine or by knock-out of an essential autophagy gene, such as Beclin 1 or ATG7, markedly reduces the ability of cells to survive during glucose deprivation. Autophagy previously was shown to degrade p62, a protein that sequesters KEAP1, and KEAP1 in turn sequesters Nrf2, a master regulator of the antioxidant response. Hence, we investigated how the Nrf2 signaling pathway might be affected by glucose deprivation and autophagy. We found that while glucose deprivation does cause decreased cellular levels of p62, Nrf2 protein levels and activity unexpectedly increase in this setting. Moreover, this increase in Nrf2 activity provides important protection to breast cancer cells during glucose deprivation, since siRNA knockdown of Nrf2 markedly impairs survival during glucose deprivation. Antioxidants, N-acetyl cysteine and glutathione also protect these cells during glucose deprivation, leading us to conclude that Nrf2 signaling via its antioxidant activity has a critical and previously undescribed role of protecting cells during glucose deprivation-induced autophagy.
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Autofagia/fisiología , Neoplasias de la Mama/metabolismo , Glucosa/deficiencia , Factor 2 Relacionado con NF-E2/metabolismo , Transducción de Señal/fisiología , Adaptación Fisiológica/fisiología , Antioxidantes/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , HumanosRESUMEN
ADIPOQ/adiponectin, an adipocytokine secreted by adipocytes in the breast tumor microenvironment, negatively regulates cancer cell growth hence increased levels of ADIPOQ/adiponectin are associated with decreased breast cancer growth. However, its mechanisms of action remain largely elusive. We report that ADIPOQ/adiponectin induces a robust accumulation of autophagosomes, increases MAP1LC3B-II/LC3B-II and decreases SQSTM1/p62 in breast cancer cells. ADIPOQ/adiponectin-treated cells and xenografts exhibit increased expression of autophagy-related proteins. LysoTracker Red-staining and tandem-mCherry-GFP-LC3B assay show that fusion of autophagosomes and lysosomes is augmented upon ADIPOQ/adiponectin treatment. ADIPOQ/adiponectin significantly inhibits breast cancer growth and induces apoptosis both in vitro and in vivo, and these events are preceded by macroautophagy/autophagy, which is integral for ADIPOQ/adiponectin-mediated cell death. Accordingly, blunting autophagosome formation, blocking autophagosome-lysosome fusion or genetic-knockout of BECN1/Beclin1 and ATG7 effectively impedes ADIPOQ/adiponectin induced growth-inhibition and apoptosis-induction. Mechanistic studies show that ADIPOQ/adiponectin reduces intracellular ATP levels and increases PRKAA1 phosphorylation leading to ULK1 activation. AMPK-inhibition abrogates ADIPOQ/adiponectin-induced ULK1-activation, LC3B-turnover and SQSTM1/p62-degradation while AMPK-activation potentiates ADIPOQ/adiponectin's effects. Further, ADIPOQ/adiponectin-mediated AMPK-activation and autophagy-induction are regulated by upstream master-kinase STK11/LKB1, which is a key node in antitumor function of ADIPOQ/adiponectin as STK11/LKB1-knockout abrogates ADIPOQ/adiponectin-mediated inhibition of breast tumorigenesis and molecular analyses of tumors corroborate in vitro mechanistic findings. ADIPOQ/adiponectin increases the efficacy of chemotherapeutic agents. Notably, high expression of ADIPOQ receptor ADIPOR2, ADIPOQ/adiponectin and BECN1 significantly correlates with increased overall survival in chemotherapy-treated breast cancer patients. Collectively, these data uncover that ADIPOQ/adiponectin induces autophagic cell death in breast cancer and provide in vitro and in vivo evidence for the integral role of STK11/LKB1-AMPK-ULK1 axis in ADIPOQ/adiponectin-mediated cytotoxic autophagy.
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Proteínas Quinasas Activadas por AMP/metabolismo , Adiponectina/farmacología , Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Autofagia/efectos de los fármacos , Neoplasias de la Mama/patología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal/efectos de los fármacos , Quinasas de la Proteína-Quinasa Activada por el AMP , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Autofagosomas/efectos de los fármacos , Autofagosomas/metabolismo , Autofagosomas/ultraestructura , Beclina-1/metabolismo , Biomarcadores/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/ultraestructura , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Metabolismo Energético/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Femenino , Humanos , Ratones , Fosforilación/efectos de los fármacosRESUMEN
Loss of function mutations in Kelch-like ECH Associated Protein 1 (KEAP1), or gain-of-function mutations in nuclear factor erythroid 2-related factor 2 (NRF2), are common in non-small cell lung cancer (NSCLC) and associated with therapeutic resistance. To discover novel NRF2 inhibitors for targeted therapy, we conducted a quantitative high-throughput screen using a diverse set of â¼400â¯000 small molecules (Molecular Libraries Small Molecule Repository Library, MLSMR) at the National Center for Advancing Translational Sciences. We identified ML385 as a probe molecule that binds to NRF2 and inhibits its downstream target gene expression. Specifically, ML385 binds to Neh1, the Cap 'N' Collar Basic Leucine Zipper (CNC-bZIP) domain of NRF2, and interferes with the binding of the V-Maf Avian Musculoaponeurotic Fibrosarcoma Oncogene Homologue G (MAFG)-NRF2 protein complex to regulatory DNA binding sequences. In clonogenic assays, when used in combination with platinum-based drugs, doxorubicin or taxol, ML385 substantially enhances cytotoxicity in NSCLC cells, as compared to single agents. ML385 shows specificity and selectivity for NSCLC cells with KEAP1 mutation, leading to gain of NRF2 function. In preclinical models of NSCLC with gain of NRF2 function, ML385 in combination with carboplatin showed significant antitumor activity. We demonstrate the discovery and validation of ML385 as a novel and specific NRF2 inhibitor and conclude that targeting NRF2 may represent a promising strategy for the treatment of advanced NSCLC.
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Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Resistencia a Antineoplásicos , Proteína 1 Asociada A ECH Tipo Kelch/genética , Neoplasias Pulmonares/tratamiento farmacológico , Factor 2 Relacionado con NF-E2/antagonistas & inhibidores , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologíaRESUMEN
AMP-activated Protein Kinase (AMPK) activity retards growth of many types of cancers. Investigating effects of AMPK activation on breast cancer cell signaling and survival, we found that breast cancer cell lines with amplification and over-expression of HER2 or EGFR are 2- to 5-fold more sensitive to cytotoxic effects of AICAR, a canonical pharmacological activator of AMPK, than breast cancer cell lines lacking HER2 or EGFR overexpression. Paralleling effects on cell survival, AICAR leads to dose- and time-dependent inhibition of HER2 and EGFR in HER2-amplified breast cancer cells, with activation of AMPK and suppression of HER2/ EGFR activity preceding commitment to cell death. Transfection of constitutively active AMPKα also leads to decreased HER2 and EGFR phosphorylation, reduced downstream signaling associated with these receptor tyrosine kinases (RTKs), and reduced breast cancer cell growth, confirming effects of AMPK activity on HER2/ EGFR. Ensuing co-immunoprecipitation experiments demonstrated an interaction of HER2 with AMPK and an in vitro phosphorylation assay found that HER2 and EGFR contain sequences that are potential substrates for AMPK. Our results lead us to postulate that AMPK regulates HER2 and EGFR activity in HER2-amplified breast cancer cells and thus activation of AMPK might provide therapeutic benefit in such cancers.
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Proteínas Quinasas Activadas por AMP/metabolismo , Neoplasias de la Mama/metabolismo , Receptores ErbB/metabolismo , Receptor ErbB-2/metabolismo , Proteínas Quinasas Activadas por AMP/genética , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacología , Animales , Western Blotting , Neoplasias de la Mama/genética , Neoplasias de la Mama/prevención & control , Línea Celular , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Humanos , Células MCF-7 , Ratones Endogámicos NOD , Ratones SCID , Interferencia de ARN , Ribonucleótidos/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
OBJECTIVE: To investigate and compare blood losses intra and postoperatively between lumbar fusion patients with and without antiplatelet use. METHODS: A total of 106 patients who had undergone at least 2 or more segments of lumbar fusion surgery were selected for the study. They were divided into three groups. Group 1 was not on medication before the surgery. Groups 2 and 3 had taken aspirin prior to the surgery. Group 2 discontinued the medication 1 week before the operation, but group 3 continued the use. In addition, non-steroid anti-inflammatory drug (NSAIDs) use in all patients was questioned. Amount of blood losses and platelet function were evaluated. RESULTS: When usage of NSAID was not controlled, intraoperative, postoperative, and total blood losses were found to have no statistical significance among the groups. However, when NSAID usage was taken into account, there were significantly higher blood losses in groups 2 and 3 compared with group 1. The use of NSAID resulted in significantly higher blood loss in group 1, but not in groups 2 or 3. The platelet function test results disclosed statistical differences between groups 1 and 2 and groups 1 and 3. CONCLUSION: Aspirin significantly increases the risk of bleeding in patients undergoing lumbar fusion at two or more levels. This risk is present even in patients who discontinued aspirin 1 week prior to surgery. In patients with high risk of complications resulting from aspirin discontinuation, the use should be allowed in lumbar fusion surgery. However, strong attention must be paid to avoid excessive bleeding. Because NSAID use also increases surgical blood loss, proper interval from discontinuation to surgery must be granted to minimize the risk.
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Antiinflamatorios no Esteroideos/efectos adversos , Aspirina/efectos adversos , Pérdida de Sangre Quirúrgica , Vértebras Lumbares/cirugía , Hemorragia Posoperatoria/inducido químicamente , Fusión Vertebral , Anciano , Pruebas de Coagulación Sanguínea/métodos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Hemorragia Posoperatoria/diagnóstico , Hemorragia Posoperatoria/etiología , Fusión Vertebral/efectos adversosRESUMEN
The efficacy of drugs used to treat cancer can be significantly attenuated by adaptive responses of neoplastic cells to drug-induced stress. To determine how cancer cells respond to inhibition of the enzyme fatty acid synthase (FAS), we focused on NF-κB-mediated pathways, which can be activated by various cellular stresses. Treating lung cancer cells with C93, a pharmacological inhibitor of FAS, results in changes indicative of a rapid initiation of NF-κB signaling, including translocation of RelA/p65 NF-κB to the nucleus, activation of a transfected NF-κB-luciferase reporter, and increased expression of NF-κB-dependent transcripts, IL-6, IL-8, and COX-2. Verifying that these responses to C93 are specifically related to inhibition of FAS, we confirmed that levels of these same transcripts increase in response to siRNA targeting FAS. Inhibiting this NF-κB response (either by transfecting a mutant IκBα or treating with bortezomib) resulted in increased cell killing by C93, indicating that the NF-κB response is protective in this setting. Because inhibiting FAS leads to accumulation of intermediate metabolites of fatty acid biosynthesis, we then questioned whether protein kinase C (PKC) is involved in this response to metabolic stress. Immunofluorescence microscopy revealed that C93 treatment results in cellular translocation of PKCα and PKCß isoforms and increased PKCα-dependent phosphorylation of the IκBα subunit of NF-κB. Furthermore, inhibiting PKC activity with RO-31-8220 or PKCα isoform-specific siRNA attenuates C93-induced IκBα phosphorylation and NF-κB activation and also potentiates C93-induced cell killing. These results suggest a link between PKC and NF-κB in protecting cancer cells from metabolic stress induced by inhibiting FAS.
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Inhibidores Enzimáticos/farmacología , Ácido Graso Sintasas/antagonistas & inhibidores , Neoplasias Pulmonares/metabolismo , FN-kappa B/fisiología , Neoplasias/metabolismo , Línea Celular Tumoral , Humanos , Neoplasias Pulmonares/patología , Neoplasias/patología , Fosforilación , Sustancias Protectoras , Proteína Quinasa CRESUMEN
Most human cancers are aneuploid and have chromosomal instability, which contrasts to the inability of human cells to normally tolerate aneuploidy. Noting that aneuploidy in human breast cancer correlates with increased expression levels of the Mps1 checkpoint gene, we investigated whether these high levels of Mps1 contribute to the ability of breast cancer cells to tolerate this aneuploidy. Reducing Mps1 levels in cultured human breast cancer cells by RNAi resulted in aberrant mitoses, induction of apoptosis, and decreased ability of human breast cancer cells to grow as xenografts in nude mice. Remarkably, breast cancer cells that survive reductions in levels of Mps1 have relatively less aneuploidy, as measured by copies of specific chromosomes, compared with cells that have constitutively high levels of Mps1. Thus, high levels of Mps1 in breast cancer cells likely contribute to these cells tolerating aneuploidy.
Asunto(s)
Aneuploidia , Neoplasias de la Mama/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Apoptosis/fisiología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proteínas de Ciclo Celular/genética , Femenino , Humanos , Ratones , Ratones Desnudos , Mitosis , Trasplante de Neoplasias , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Quinasas , Interferencia de ARN , Células Tumorales CultivadasRESUMEN
A pharmaceutical grade extract of Coix lachryma-jobi seeds is currently the most commonly used treatment for cancer in China. Although clinical data support the use of this preparation of a Traditional Chinese Medicine for cancer treatment, biological basis for the activity of this preparation has not been previously established. To address this issue, we first evaluated the anti-neoplastic activity of a Coix extract emulsion in xenografts of MDA-MB-231 breast cancer cells and found that the extract significantly inhibits growth of MDA-MB-231 xenografts in athymic nude mice. Using oligonucleotide microarrays, we determined that Coix seed extract also significantly affects gene expression in these cells, including downregulation of genes (such as COX-2 and matrixmetalloproteinases) that are considered to be important in neoplasia. The specific gene expression changes noted after Coix seed extract treatment are characteristic of inhibition of NFkappaB-dependent transcription, leading us to evaluate how the treatment affects that pathway. An NFkappaB-dependent reporter assay demonstrated dose-dependant inhibition of NFkappaB signaling by treatment of cultures with the extract, and immunofluorescent microscopy found that these effects are associated with reduced translocation of the Rel-A/p65 subunit of NFkappaB to the nucleus. Coix extract also inhibits activity of protein kinase C, a major mediator of signal transduction and activator of NFkappaB. Thus, this Traditional Chinese Medicine-based cancer treatment affects cellular pathways of recognized importance in neoplasia.
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Antineoplásicos Fitogénicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Coix/química , Medicamentos Herbarios Chinos/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Proteínas de Neoplasias/antagonistas & inhibidores , Fitoterapia , Proteína Quinasa C/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Factor de Transcripción ReIA/antagonistas & inhibidores , Transporte Activo de Núcleo Celular/efectos de los fármacos , Adenocarcinoma/patología , Animales , Antineoplásicos Fitogénicos/uso terapéutico , Neoplasias de la Mama/patología , Línea Celular Tumoral/efectos de los fármacos , Línea Celular Tumoral/trasplante , Relación Dosis-Respuesta a Droga , Medicamentos Herbarios Chinos/uso terapéutico , Emulsiones , Femenino , Humanos , Neoplasias Pulmonares/patología , Ratones , Ratones Desnudos , Invasividad Neoplásica , Proteínas de Neoplasias/metabolismo , Semillas/química , Factor de Transcripción ReIA/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: Most breast cancers have chromosomal instability that seems related to defective mitotic spindle checkpoints. Because the molecular basis of this defect is unknown, we evaluated breast cancer cell lines and tissues for possible defects involving the major mitotic checkpoint genes responsible for maintaining chromosomal stability. EXPERIMENTAL DESIGN: We analyzed sequences and expression levels (RNA and protein) of eight major spindle checkpoint genes (MAD1L1, MAD2L1, MAD2L2, BUB1, BUB1B, BUB3, CDC20, and TTK) in a panel of 12 breast cancer cell lines, most with established genetic instability and defective spindle damage checkpoint response. mRNA levels of these genes were also measured in primary tumor samples, and immunohistochemical staining was used to evaluate BUB1B protein levels in a panel of 270 additional cases of breast cancer. RESULTS: No functionally significant sequence variations were found for any of the eight genes in the breast cancer cell lines with chromosomal instability. More surprisingly, the mRNA and protein levels for these checkpoint genes are significantly higher in the genetically unstable breast cancer cell lines and in high-grade primary breast cancer tissues than in the stable (and checkpoint proficient) MCF-10A and normal mammary epithelial cells, or in normal breast tissues. In fact, overexpression of the BUB1B protein is a marker that recognizes nearly 80% of breast cancers in paraffin-embedded tissues. CONCLUSIONS: Defective mitotic spindle checkpoints in breast cancer are most likely not caused by low expression or mutations of these eight checkpoint genes. High levels of these particular transcripts could represent a cellular compensation for defects in other molecular components of the mitotic spindle damage checkpoint, and increased expression of these genes might be markers of breast cancers with chromosomal instability.
Asunto(s)
Neoplasias de la Mama/genética , Regulación Neoplásica de la Expresión Génica , Proteínas de Neoplasias/genética , Proteínas Quinasas/genética , Huso Acromático/genética , Adenocarcinoma Mucinoso/genética , Adenocarcinoma Mucinoso/metabolismo , Adenocarcinoma Mucinoso/patología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patología , Carcinoma Papilar/genética , Carcinoma Papilar/metabolismo , Carcinoma Papilar/patología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Fragilidad Cromosómica , Femenino , Variación Genética , Humanos , Mitosis/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales CultivadasRESUMEN
Proteolytic degradation of the extracellular matrix and tumor metastasis correlate with the expression of endopeptidases known as matrix metalloproteinases (MMPs). The expression of MMPs is regulated by cytokines and signal transduction pathways, including those activated by phorbol myristate acetate (PMA). We found that resveratrol, a phytoalexin present in grapes, significantly inhibits the PMA-induced increase in MMP-9 expression and activity. These effects of resveratrol are dose dependent and correlate with the suppression of MMP-9 mRNA expression levels. PMA caused about a 23-fold increase in MMP-9 promoter activity, which was suppressed by resveratrol. Transient transfection utilizing MMP-9 constructs, in which specific transcriptional factors were mutagenized, indicated that the effects of PMA and resveratrol were mediated via an activator protein-1 and nuclear factor-kappaB response element. Resveratrol inhibited PMA-mediated activation of c-Jun N-terminal kinase (JNK) and protein kinase C (PKC)-delta activation. Therefore, we conclude that the MMP-9 inhibition activity of resveratrol and its inhibition of JNK and PKC-delta may have a therapeutic potential, given that a novel means of controlling growth and invasiveness of tumors.
Asunto(s)
Antineoplásicos Fitogénicos/toxicidad , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Proteínas Quinasas JNK Activadas por Mitógenos , Metaloproteinasa 9 de la Matriz/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Proteína Quinasa C/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Estilbenos/toxicidad , Acetato de Tetradecanoilforbol/antagonistas & inhibidores , Secuencia de Bases , Línea Celular Tumoral , Clonación Molecular , Cartilla de ADN , Femenino , Humanos , MAP Quinasa Quinasa 4 , FN-kappa B/metabolismo , Plásmidos , Regiones Promotoras Genéticas , Proteína Quinasa C-delta , Resveratrol , Acetato de Tetradecanoilforbol/toxicidad , Factor de Transcripción AP-1/metabolismo , Transfección , Neoplasias del Cuello UterinoRESUMEN
Proteolytic degradation of the extracellular matrix and tumor metastasis correlate with expression of endopeptidases known as matrix metalloproteinases (MMPs). Expression of MMPs is regulated by cytokines and signal transduction pathways, including those activated by phorbol myristate acetate. We found that dykellic acid, a fungal metabolite, significantly inhibits the phorbol myristate acetate-induced increase in MMP-9 expression and activity. These effects of dykellic acid are time- and dose-dependent, and correlate with decreased MMP-9 promoter activity and mRNA expression. Whereas this compound does not affect DNA binding activity of nuclear factor kappa B (NF kappa B), dykellic acid does inhibit transactivation of NF kappa B. These data demonstrate a role for NF kappa B in the regulation of MMP-9 expression and the ability of dykellic acid to suppress this action of NF kappa B.