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In cancer therapy, DNA intercalators are mainly known for their capacity to kill cells by inducing DNA damage. Recently, several DNA intercalators have attracted much interest given their ability to inhibit RNA Polymerase I transcription (BMH-21), evict histones (Aclarubicin) or induce chromatin trapping of FACT (Curaxin CBL0137). Interestingly, these DNA intercalators lack the capacity to induce DNA damage while still retaining cytotoxic effects and stabilize p53. Herein, we report that these DNA intercalators impact chromatin biology by interfering with the chromatin stability of RNA polymerases I, II and III. These three compounds have the capacity to induce degradation of RNA polymerase II and they simultaneously enable the trapping of Topoisomerases TOP2A and TOP2B on the chromatin. In addition, BMH-21 also acts as a catalytic inhibitor of Topoisomerase II, resembling Aclarubicin. Moreover, BMH-21 induces chromatin trapping of the histone chaperone FACT and propels accumulation of Z-DNA and histone eviction, similarly to Aclarubicin and CBL0137. These DNA intercalators have a cumulative impact on general transcription machinery by inducing accumulation of topological defects and impacting nuclear chromatin. Therefore, their cytotoxic capabilities may be the result of compounding deleterious effects on chromatin homeostasis.
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Cromatina , ADN-Topoisomerasas de Tipo II , Sustancias Intercalantes , ARN Polimerasa II , Humanos , Antígenos de Neoplasias/metabolismo , Antígenos de Neoplasias/genética , Carbazoles , Cromatina/metabolismo , Dicetopiperazinas , ADN/metabolismo , ADN/química , Daño del ADN , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas del Grupo de Alta Movilidad/metabolismo , Proteínas del Grupo de Alta Movilidad/genética , Histonas/metabolismo , Sustancias Intercalantes/farmacología , Sustancias Intercalantes/química , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/genética , ARN Polimerasa I/metabolismo , ARN Polimerasa I/antagonistas & inhibidores , ARN Polimerasa II/metabolismo , ARN Polimerasa III/metabolismo , Inhibidores de Topoisomerasa II/farmacología , Transcripción Genética/efectos de los fármacos , Factores de Elongación Transcripcional/metabolismo , Factores de Elongación Transcripcional/genética , Aclarubicina/farmacologíaRESUMEN
Drug repurposing is a versatile strategy to improve current therapies. Disulfiram has long been used in the treatment of alcohol dependency and multiple clinical trials to evaluate its clinical value in oncology are ongoing. We have recently reported that the disulfiram metabolite diethyldithiocarbamate, when combined with copper (CuET), targets the NPL4 adapter of the p97VCP segregase to suppress the growth of a spectrum of cancer cell lines and xenograft models in vivo. CuET induces proteotoxic stress and genotoxic effects, however important issues concerning the full range of the CuET-evoked tumor cell phenotypes, their temporal order, and mechanistic basis have remained largely unexplored. Here, we have addressed these outstanding questions and show that in diverse human cancer cell models, CuET causes a very early translational arrest through the integrated stress response (ISR), later followed by features of nucleolar stress. Furthermore, we report that CuET entraps p53 in NPL4-rich aggregates leading to elevated p53 protein and its functional inhibition, consistent with the possibility of CuET-triggered cell death being p53-independent. Our transcriptomics profiling revealed activation of pro-survival adaptive pathways of ribosomal biogenesis (RiBi) and autophagy upon prolonged exposure to CuET, indicating potential feedback responses to CuET treatment. The latter concept was validated here by simultaneous pharmacological inhibition of RiBi and/or autophagy that further enhanced CuET's tumor cytotoxicity, using both cell culture and zebrafish in vivo preclinical models. Overall, these findings expand the mechanistic repertoire of CuET's anti-cancer activity, inform about the temporal order of responses and identify an unorthodox new mechanism of targeting p53. Our results are discussed in light of cancer-associated endogenous stresses as exploitable tumor vulnerabilities and may inspire future clinical applications of CuET in oncology, including combinatorial treatments and focus on potential advantages of using certain validated drug metabolites, rather than old, approved drugs with their, often complex, metabolic profiles.
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Disulfiram , Neoplasias , Animales , Humanos , Línea Celular Tumoral , Disulfiram/metabolismo , Neoplasias/metabolismo , Ribosomas/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Pez Cebra/metabolismoRESUMEN
Introduction: Chronic wounds represent a frequent cause of consultation for plastic and reconstructive surgeons. The use of epidermal culture stands out because they provide complete epithelialization, adequate aesthetic-functional results, and no morbidity for the patient. Epifast® is a pre-manufactured cultured epidermal allograft derived from the amplification in vitro of human keratinocytes. Materials and Methods: A prospective longitudinal multicenter study was carried out in four chronic wound reference centers, which were in charge of plastic and reconstructive surgery services. For a standardized wound bed preparation, the protocol synthesized by the acronym "TIME" was used. At the end of the "TIME" protocol, the pre-fabricated allograft was applied and removed 7 days after its application. Results: A total of 133 patients with diagnosis of chronic wound were included in the study. The median age was 69.3 ± 13.6 years. The most common comorbidity found was diabetes mellitus type 2 in 71.4% of the patients (n = 95) and systemic arterial hypertension in 60.2% of the patients (n = 80). The most frequent location of chronic wounds was seen in the lower extremity with 45.1% (n = 60). The mean duration for it to close was 46 ± 14 days, in which they closed within the first 3 months in 93% (n = 125) of the cases. About 91.7% (n = 122) of the wounds achieved total closure. Conclusion: Cultured epidermal allograft, combined with a meticulous technique and an adequate selection of patients, represents a safe and effective tool for chronic wounds.
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Triple-negative breast cancer (TNBC) is a subtype of breast cancer that comprises various disease entities, all of which share a set of common features: a lack of expression of the estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2, respectively. Because of their receptor status, conventional chemotherapy remains the main therapeutic option for TNBC patients. We employed a reverse phase protein array approach (RPPA), complemented by immunohistochemistry, to quantitatively profile the activation state of 84 actionable key signaling intermediates and phosphoproteins in a set of 44 TNBC samples. We performed supervised and unsupervised approaches to proteomic data analysis to identify groups of samples sharing common characteristics that could be amenable to existing therapies. We found the heterogenous activation of multiple pathways, with PI3 K/AKT/mTOR signaling being the most common event. Some specific individualized therapeutic possibilities include the expression of oncogenic KIT in association with cytokeratin 15 and Erk1/2 positive tumors, both of which may have clinical value.
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Proteómica , Neoplasias de la Mama Triple Negativas/metabolismo , Carcinogénesis/patología , Humanos , Proteínas de Neoplasias/metabolismo , Fosforilación , Análisis por Matrices de Proteínas , Proteínas Proto-Oncogénicas c-kit/genética , Transducción de SeñalRESUMEN
Eukaryotic initiation factor 4A-III (eIF4A3), a core helicase component of the exon junction complex, is essential for splicing, mRNA trafficking, and nonsense-mediated decay processes emerging as targets in cancer therapy. Here, we unravel eIF4A3's tumor-promoting function by demonstrating its role in ribosome biogenesis (RiBi) and p53 (de)regulation. Mechanistically, eIF4A3 resides in nucleoli within the small subunit processome and regulates rRNA processing via R-loop clearance. EIF4A3 depletion induces cell cycle arrest through impaired RiBi checkpoint-mediated p53 induction and reprogrammed translation of cell cycle regulators. Multilevel omics analysis following eIF4A3 depletion pinpoints pathways of cell death regulation and translation of alternative mouse double minute homolog 2 (MDM2) transcript isoforms that control p53. EIF4A3 expression and subnuclear localization among clinical cancer specimens correlate with the RiBi status rendering eIF4A3 an exploitable vulnerability in high-RiBi tumors. We propose a concept of eIF4A3's unexpected role in RiBi, with implications for cancer pathogenesis and treatment.
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ARN Helicasas DEAD-box , Proteína p53 Supresora de Tumor , Animales , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , ADN Helicasas/metabolismo , Factor 4A Eucariótico de Iniciación/genética , Factor 4A Eucariótico de Iniciación/metabolismo , Exones/genética , Ratones , Ribosomas/genética , Ribosomas/metabolismo , Proteína p53 Supresora de Tumor/genéticaRESUMEN
OBJETIVO: Evaluar la exactitud diagnóstica del Índice de Riesgo de Malignidad II (IRM II) en 100 pacientes con diagnóstico de masa anexial. MÉTODO: En una muestra de 100 pacientes con diagnóstico de masa anexial se cuantificaron variables demográficas y se aplicó el IRM II. Mediante una tabla 2 × 2 se obtuvieron la sensibilidad, la especificidad, el valor predictivo positivo, el valor predictivo negativo y la exactitud diagnóstica, y se compararon con el resultado histopatológico. RESULTADOS: El IRM II con resultado positivo se presentó en el 73.1% (52 pacientes) de los casos con resultado histopatológico maligno y en el 26.9% de aquellos con resultado histopatológico benigno. Presenta una sensibilidad en la prueba del 73.1%, una especificidad del 70.8%, un valor predictivo positivo del 73.2% y un valor predictivo negativo del 70.8%. La exactitud diagnóstica es del 72%. CONCLUSIONES: El IRM II es una herramienta de cribaje con aceptable desempeño diagnóstico para normar la conducta, como referir a un centro especializado o solicitar estudios más específicos en pacientes con diagnóstico de masa anexial por sospecha de malignidad. OBJECTIVE: Evaluate the usefulness of the Malignancy Risk Index II (MRI II) using diagnostic accuracy variables in 100 patients diagnosed with adnexal mass. METHOD: In a sample of 100 patients with a diagnosis of adnexal mass, demographic variables were quantified and MRI II was applied. The sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic accuracy were obtained using a 2 × 2 table and compared with the histopathological result. RESULTS: MRI II with positive result was presented in 73.1% (52 patients) with malignant histopathological result and in 26.9% of patients with benign histopathological result. It presents a sensitivity in the test of 73.1%, a specificity of 70.8%, positive and negative predictive value of 73.2 and 70.8%, respectively. Diagnostic accuracy of 72%. CONCLUSIONS: MRI II is a screening tool with acceptable diagnostic performance to regulate behavior, such as referring to a specialized center or requesting more specific studies in patients diagnosed with adnexal mass due to suspected malignancy.
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Hospitales , Humanos , Estudios RetrospectivosRESUMEN
Gallbladder stones (cholecystolithiasis) are the main risk factor for gallbladder cancer (GBC), a lethal biliary malignancy with poor survival rates worldwide. Gallbladder stones are thought to damage the gallbladder epithelium and trigger chronic inflammation. Preneoplastic lesions that arise in such an inflammatory microenvironment can eventually develop into invasive carcinoma, through mechanisms that are not fully understood. Here, we developed a novel gallbladder preneoplasia mouse model through the administration of two lithogenic diets (a low- or a high-cholesterol diet) in wild-type C57BL/6 mice over a period of 9 months. Additionally, we evaluated the chemopreventive potentials of the anti-inflammatory drug aspirin and the cholesterol absorption inhibitor ezetimibe. Both lithogenic diets induced early formation of gallbladder stones, together with extensive inflammatory changes and widespread induction of metaplasia, an epithelial adaptation to tissue injury. Dysplastic lesions were presented only in mice fed with high-cholesterol diet (62.5%) in late stages (9th month), and no invasive carcinoma was observed at any stage. The cholesterol absorption inhibitor ezetimibe inhibited gallbladder stone formation and completely prevented the onset of metaplasia and dysplasia in both lithogenic diets, whereas aspirin partially reduced metaplasia development only in the low-cholesterol diet setting. This model recapitulates several of the structural and inflammatory findings observed in human cholecystolithiasic gallbladders, making it relevant for the study of gallbladder carcinogenesis. In addition, our results suggest that the use of cholesterol absorption inhibitors and anti-inflammatory drugs can be evaluated as chemopreventive strategies to reduce the burden of GBC among high-risk populations.
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Aspirina/uso terapéutico , Quimioprevención , Ezetimiba/uso terapéutico , Neoplasias de la Vesícula Biliar/tratamiento farmacológico , Neoplasias de la Vesícula Biliar/prevención & control , Lesiones Precancerosas/tratamiento farmacológico , Lesiones Precancerosas/prevención & control , Animales , Colecistolitiasis/complicaciones , Colesterol/metabolismo , Colesterol en la Dieta , Enfermedad Crónica , Dieta , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Hígado Graso/patología , Conducta Alimentaria , Neoplasias de la Vesícula Biliar/patología , Cálculos Biliares/etiología , Cálculos Biliares/patología , Inflamación/patología , Masculino , Metaplasia , Ratones Endogámicos C57BL , Lesiones Precancerosas/patología , Bazo/patologíaRESUMEN
BACKGROUND: Gallbladder cancer (GBC) is the most common tumor of the biliary tract. The incidence of GBC shows a large geographic variability, being particularly frequent in Native American populations. In Chile, GBC represents the second cause of cancer-related death among women. We describe here the establishment of three novel cell lines derived from the ascitic fluid of a Chilean GBC patient, who presented 46% European, 36% Mapuche, 12% Aymara and 6% African ancestry. RESULTS: After immunocytochemical staining of the primary cell culture, we isolated and comprehensively characterized three independent clones (PUC-GBC1, PUC-GBC2 and PUC-GBC3) by short tandem repeat DNA profiling and RNA sequencing as well as karyotype, doubling time, chemosensitivity, in vitro migration capability and in vivo tumorigenicity assay. Primary culture cells showed high expression of CK7, CK19, CA 19-9, MUC1 and MUC16, and negative expression of mesothelial markers. The three isolated clones displayed an epithelial phenotype and an abnormal structure and number of chromosomes. RNA sequencing confirmed the increased expression of cytokeratin and mucin genes, and also of TP53 and ERBB2 with some differences among the three cells lines, and revealed a novel exonic mutation in NF1. The PUC-GBC3 clone was the most aggressive according to histopathological features and the tumorigenic capacity in NSG mice. CONCLUSIONS: The first cell lines established from a Chilean GBC patient represent a new model for studying GBC in patients of Native American descent.
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Antígenos de Carbohidratos Asociados a Tumores/genética , Neoplasias de la Vesícula Biliar/genética , Indígenas Sudamericanos/genética , Animales , Antineoplásicos/farmacología , Líquido Ascítico/metabolismo , Carcinogénesis/genética , Pruebas de Carcinogenicidad , Línea Celular Tumoral/efectos de los fármacos , Línea Celular Tumoral/metabolismo , Chile , Cisplatino/farmacología , Células Clonales/efectos de los fármacos , Células Clonales/metabolismo , Dermatoglifia del ADN , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Células Epiteliales/metabolismo , Neoplasias de la Vesícula Biliar/metabolismo , Perfilación de la Expresión Génica , Genes erbB-2/genética , Humanos , Queratina-19/genética , Queratina-7/genética , Masculino , Ratones Endogámicos NOD , Persona de Mediana Edad , Receptor ErbB-2/genética , Análisis de Secuencia de ARN , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/genética , GemcitabinaRESUMEN
Gallbladder cancer is an aggressive disease with late diagnosis and no efficacious treatment. The Hippo-Yes-associated protein 1 (YAP1) signaling pathway has emerged as a target for the development of new therapeutic interventions in cancers. However, the role of the Hippo-targeted therapy has not been addressed in advanced gallbladder cancer (GBC). This study aimed to evaluate the expression of the major Hippo pathway components mammalian Ste20-like protein kinase 1 (MST1), YAP1 and transcriptional coactivator with PDZ-binding motif (TAZ) and examined the effects of Verteporfin (VP), a small molecular inhibitor of YAP1-TEA domain transcription factor (TEAD) protein interaction, in metastatic GBC cell lines and patient-derived organoids (PDOs). Immunohistochemical analysis revealed that advanced GBC patients had high nuclear expression of YAP1. High nuclear expression of YAP1 was associated with poor survival in GBC patients with subserosal invasion (pT2). Additionally, advanced GBC cases showed reduced expression of MST1 compared to chronic cholecystitis. Both VP treatment and YAP1 siRNA inhibited the migration ability in GBC cell lines. Interestingly, gemcitabine resistant PDOs with high nuclear expression of YAP1 were sensitive to VP treatment. Taken together, our results suggest that key components of the Hippo-YAP1 signaling pathway are dysregulated in advanced gallbladder cancer and reveal that the inhibition YAP1 may be a candidate for targeted therapy.
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Pharmacological inhibition of ribosome biogenesis is a promising avenue for cancer therapy. Herein, we report a novel activity of the FDA-approved antimalarial drug amodiaquine which inhibits rRNA transcription, a rate-limiting step for ribosome biogenesis, in a dose-dependent manner. Amodiaquine triggers degradation of the catalytic subunit of RNA polymerase I (Pol I), with ensuing RPL5/RPL11-dependent stabilization of p53. Pol I shutdown occurs in the absence of DNA damage and without the subsequent ATM-dependent inhibition of rRNA transcription. RNAseq analysis revealed mechanistic similarities of amodiaquine with BMH-21, the first-in-class Pol I inhibitor, and with chloroquine, the antimalarial analog of amodiaquine, with well-established autophagy-inhibitory activity. Interestingly, autophagy inhibition caused by amodiaquine is not involved in the inhibition of rRNA transcription, suggesting two independent anticancer mechanisms. In vitro, amodiaquine is more efficient than chloroquine in restraining the proliferation of human cell lines derived from colorectal carcinomas, a cancer type with predicted susceptibility to ribosome biogenesis stress. Taken together, our data reveal an unsuspected activity of a drug approved and used in the clinics for over 30 years, and provide rationale for repurposing amodiaquine in cancer therapy.
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Amodiaquina/farmacología , Antimaláricos/farmacología , Antineoplásicos/farmacología , Autofagia/efectos de los fármacos , Neoplasias Colorrectales/tratamiento farmacológico , Ribosomas/efectos de los fármacos , Proteína p53 Supresora de Tumor/metabolismo , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Neoplasias Colorrectales/diagnóstico por imagen , Neoplasias Colorrectales/metabolismo , Humanos , Imagen Óptica , Ribosomas/genética , Ribosomas/metabolismo , Transcripción Genética/efectos de los fármacos , Transcripción Genética/genética , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/genéticaRESUMEN
BACKGROUND: Gallbladder cancer (GBC) is the most common tumor of the biliary tract. The incidence of GBC shows a large geographic variability, being particularly frequent in Native American populations. In Chile, GBC represents the second cause of cancer-related death among women. We describe here the establishment of three novel cell lines derived from the ascitic fluid of a Chilean GBC patient, who presented 46% European, 36% Mapuche, 12% Aymara and 6% African ancestry. RESULTS: After immunocytochemical staining of the primary cell culture, we isolated and comprehensively characterized three independent clones (PUC-GBC1, PUC-GBC2 and PUC-GBC3) by short tandem repeat DNA profiling and RNA sequencing as well as karyotype, doubling time, chemosensitivity, in vitro migration capability and in vivo tumorigenicity assay. Primary culture cells showed high expression of CK7, CK19, CA 19-9, MUC1 and MUC16, and negative expression of mesothelial markers. The three isolated clones displayed an epithelial phenotype and an abnormal structure and number of chromosomes. RNA sequencing confirmed the increased expression of cytokeratin and mucin genes, and also of TP53 and ERBB2 with some differences among the three cells lines, and revealed a novel exonic mutation in NF1. The PUC-GBC3 clone was the most aggressive according to histopathological features and the tumorigenic capacity in NSG mice. CONCLUSIONS: The first cell lines established from a Chilean GBC patient represent a new model for studying GBC in patients of Native American descent.
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Humanos , Animales , Masculino , Persona de Mediana Edad , Antígenos de Carbohidratos Asociados a Tumores/genética , Indígenas Sudamericanos/genética , Neoplasias de la Vesícula Biliar/genética , Líquido Ascítico/metabolismo , Células Tumorales Cultivadas , Pruebas de Carcinogenicidad , Chile , Dermatoglifia del ADN , Proteína p53 Supresora de Tumor/genética , Cisplatino/farmacología , Ratones Endogámicos NOD , Células Clonales/efectos de los fármacos , Células Clonales/metabolismo , Análisis de Secuencia de ARN , Receptor ErbB-2/genética , Genes erbB-2/genética , Perfilación de la Expresión Génica , Línea Celular Tumoral/efectos de los fármacos , Línea Celular Tumoral/metabolismo , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Células Epiteliales/metabolismo , Queratina-19/genética , Queratina-7/genética , Carcinogénesis/genética , Neoplasias de la Vesícula Biliar/metabolismo , Antineoplásicos/farmacologíaRESUMEN
Cancers elicit an immune response by modifying the microenvironment. The immune system plays a pivotal role in cancer recognition and eradication. While the potential clinical value of infiltrating lymphocytes at the tumor site has been assessed in breast cancer, circulating cytokines - the molecules coordinating and fine-tuning immune response - are still poorly characterized. Using two breast cancer cohorts (MicMa, n = 131, DCTB, n = 28) and the multiplex Luminex platform, we measured the levels of 27 cytokines in the serum of breast cancer patients prior to treatment. We investigated the cytokine levels in relation to clinicopathological characteristics and in perspective of the tumor infiltrating immune cells predicted from the bulk mRNA expression data. Unsupervised clustering analysis of the serum cytokine levels in the MicMa cohort identified a cluster of pro-inflammatory, pro-angiogenic, and Th2-related cytokines which was associated with poor prognosis. Notably high levels of platelet derived growth factor BB (PDGF) reflected a more aggressive tumor phenotype and larger tumor size. A significant positive correlation between serum levels of interferon gamma-induced protein 10 (IP10) and its mRNA expression at the tumor site suggested that tumor-IP10-production may outflow to the bloodstream. High IP10 serum levels were associated with a worse prognosis. Finally, we found serum levels of both PDGF and IP10 associated with enrichment scores of specific tumor infiltrating immune cells. Our study suggests that monitoring cytokine circulating levels in breast cancer could be used to characterize breast cancers and the immune composition of their microenvironment through readily available biological material.
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AIMS: Gallbladder cancer (GBC) is an aggressive tumour that is usually diagnosed at advanced stages and is characterised by a poor prognosis. Using public data of normal human tissues, we found that mRNA and protein levels of mucin 5B (MUC5B) and carbonic anhydrase 9 (CA9) were highly increased in gallbladder tissues. In addition, previous evidence has shown that claudin 18 (CLDN18) protein expression is higher in GBC. The aim of this study was to perform an analysis of these cell surface proteins during the histological progression of GBC in order to identify their theranostic potential. METHODS AND RESULTS: MUC5B expression, CA9 expression and CLDN18 expression were examined by immunohistochemistry in a series of 179 chronic cholecystitis (including 16 metaplastic tissues), 15 dysplasia and 217 GBC samples by the use of tissue microarray analysis. A composite staining score was calculated from staining intensity and percentage of positive cells. Immunohistochemical analysis showed high expression of MUC5B and CA9 among normal epithelium, metaplastic tissues, and dysplastic tissues. However, expression of both proteins was observed in roughly 50% of GBC samples. In contrast, CLDN18 was absent in normal epithelium, but its expression was higher in metaplastic cells. Among GBC cases, approximately half showed high CLDN18 expression. No associations were found between MUC5B, CA9 and CLDN18 expression and any clinicopathological features. CONCLUSIONS: CLDN18 is a new metaplasia marker in gallbladder tissues, and is conserved in approximately half of GBC cases. MUC5B and CA9 are highly conserved during GBC histological progression. The three markers are potential theranostic markers, in particular CA9 and CLDN18, for which there are already targeted therapies available.
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Antígenos de Neoplasias/biosíntesis , Biomarcadores de Tumor/análisis , Anhidrasa Carbónica IX/biosíntesis , Claudinas/biosíntesis , Neoplasias de la Vesícula Biliar/patología , Mucina 5B/biosíntesis , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Nanomedicina Teranóstica/métodosRESUMEN
The homeodomain transcription factor PROX1 has been linked to several cancer types, including gliomas, but its functions remain to be further elucidated. Here we describe a functional role and the prognostic value of PROX1 in glioblastoma. Low expression of PROX1 correlated with poor overall survival and the mesenchymal glioblastoma subtype signature. The latter finding was recapitulated in vitro, where suppression or overexpression of PROX1 in glioma cell cultures transitioned cells to a mesenchymal or to a nonmesenchymal glioblastoma gene expression signature, respectively. PROX1 modulation affected proliferation rates that coincided with changes in protein levels of CCNA1 and CCNE1 as well as the cyclin inhibitors CDKN1A, CDKN1B, and CDKN1C. Overexpression of SOX2 increased PROX1 expression, but treatment with a CDK2 inhibitor subsequently decreased PROX1 expression, which was paralleled by decreased SOX2 levels. The THRAP3 protein was a novel binding partner for PROX1, and suppression of THRAP3 increased both transcript and protein levels of PROX1. Together, these findings highlight the prognostic value of PROX1 and its role as a regulator of glioblastoma gene expression subtypes, intratumoral heterogeneity, proliferation, and cell-cycle control.Significance: These findings demonstrate the role and prognostic value of PROX1 in glioblastomas; low PROX1 levels correlate with a mesenchymal gene expression subtype and shorter survival in glioblastoma tumors. Cancer Res; 78(20); 5901-16. ©2018 AACR.
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Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Glioblastoma/metabolismo , Proteínas de Homeodominio/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Proteínas de Unión al ADN/metabolismo , Supervivencia sin Enfermedad , Glioma/metabolismo , Humanos , Espectrometría de Masas , Mesodermo/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Trasplante de Neoplasias , Pronóstico , ARN Interferente Pequeño/metabolismo , Factores de Transcripción SOXB1/metabolismo , Factores de Transcripción/metabolismo , Resultado del TratamientoRESUMEN
Gallbladder cancer (GBC) is a lethal cancer with poor prognosis associated with high invasiveness and poor response to chemotherapy and radiotherapy. New therapeutic approaches are urgently needed in order to improve survival and response rates of GBC patients. We screened 130 small molecule inhibitors on a panel of seven GBC cell lines and identified the HSP90 inhibitor 17-AAG as one of the most potent inhibitory drugs across the different lines. We tested the antitumor efficacy of 17-AAG and geldanamycin (GA) in vitro and in a subcutaneous preclinical tumor model NOD-SCID mice. We also evaluated the expression of HSP90 by immunohistochemistry in human GBC tumors.In vitro assays showed that 17-AAG and GA significantly reduced the expression of HSP90 target proteins, including EGFR, AKT, phospho-AKT, Cyclin B1, phospho-ERK and Cyclin D1. These molecular changes were consistent with reduced cell viability and cell migration and promotion of G2/M cell cycle arrest and apoptosis observed in our in vitro studies.In vivo, 17-AAG showed efficacy in reducing subcutaneous tumors size, exhibiting a 69.6% reduction in tumor size in the treatment group compared to control mice (p < 0.05).The HSP90 immunohistochemical staining was seen in 182/209 cases of GBC (87%) and it was strongly expressed in 70 cases (33%), moderately in 58 cases (28%), and weakly in 54 cases (26%).Our pre-clinical observations strongly suggest that the inhibition of HSP90 function by HSP90 inhibitors is a promising therapeutic strategy for gallbladder cancer that may benefit from new HSP90 inhibitors currently in development.
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Antineoplásicos/farmacología , Benzoquinonas/farmacología , Descubrimiento de Drogas , Ensayos de Selección de Medicamentos Antitumorales , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Lactamas Macrocíclicas/farmacología , Animales , Apoptosis/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Descubrimiento de Drogas/métodos , Ensayos de Selección de Medicamentos Antitumorales/métodos , Neoplasias de la Vesícula Biliar , Ensayos Analíticos de Alto Rendimiento , Humanos , Ratones , Bibliotecas de Moléculas Pequeñas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Gallbladder cancer (GBC) is an aggressive malignancy. Although surgical resection may be curable, most patients are diagnosed at an advanced unresectable disease stage. Cholelithiasis is the major risk factor; however the pathogenesis of the disease, from gallstone cholecystitis to cancer, is still not understood. To understand the molecular genetic underpinnings of this cancer and explore novel therapeutic targets for GBC, we examined the key genes and pathways involved in GBC using RNA sequencing. We performed gene expression analysis of 32 cases of surgically-resected GBC along with normal gallbladder tissue controls. We observed that 519 genes were differentially expressed between GBC and normal GB mucosal controls. The liver X receptor (LXR)/retinoid X receptor (RXR) and farnesoid X receptor (FXR) /RXR pathways were the top canonical pathways involved in GBC. Key genes in these pathways, including SERPINB3 and KLK1, were overexpressed in GBC, especially in female GBC patients. Additionally, ApoA1 gene expression suppressed in GBC as compared with normal control tissues. LXR and FXR genes, known to be important in lipid metabolism also function as tumor suppressors and their down regulation appears to be critical for GBC pathogenesis. LXR agonists may have therapeutic value and as potential therapeutic targets.
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Neoplasias de la Vesícula Biliar/metabolismo , Metabolismo de los Lípidos/fisiología , Receptores X del Hígado/metabolismo , Anciano , Anciano de 80 o más Años , Femenino , Neoplasias de la Vesícula Biliar/patología , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Persona de Mediana Edad , Receptores Citoplasmáticos y Nucleares/metabolismoRESUMEN
The PI3K/AKT/mTOR pathway plays a crucial role in the regulation of multiple cellular functions including cell growth, proliferation, metabolism and angiogenesis. Emerging evidence has shown that deregulation of this pathway has a role promoting gastric cancer (GC). The aim was to assess the expression of genes involved in this pathway by qPCR in 23 tumor and 23 non-tumor gastric mucosa samples from advanced GC patients, and in AGS, MKN28 and MKN45 gastric cancer cell lines. Results showed a slight overexpression of PIK3CA, PIK3CB, AKT1, MTOR, RPS6KB1, EIF4EBP1 and EIF4E genes, and a slightly decreased PTEN and TSC1 expression. In AGS, MKN28 and MKN45 cells a significant gene overexpression of PIK3CA, PIK3CB, AKT1, MTOR, RPS6KB1 and EIF4E, and a significant repression of PTEN gene expression were observed. Immunoblotting showed that PI3K-ß, AKT, p-AKT, PTEN, mTOR, p-mTOR, P70S6K1, p-P70S6K1, 4E-BP1, p-4E-BP1, eIF4E and p-eIF4E proteins were present in cell lines at different levels, confirming activation of this pathway in vitro. This is the first time this extensive panel of 9 genes within PI3K/AKT/mTOR pathway has been studied in GC to clarify the biological role of this pathway in GC and develop new strategies for this malignancy.
Asunto(s)
Expresión Génica/genética , Fosfatidilinositol 3-Quinasas/genética , Proteínas Proto-Oncogénicas c-akt/genética , Neoplasias Gástricas/genética , Serina-Treonina Quinasas TOR/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas de Ciclo Celular , Línea Celular Tumoral , Fosfatidilinositol 3-Quinasa Clase I , Humanos , Fosfohidrolasa PTEN/genética , Fosfoproteínas/genética , Proteínas Quinasas S6 Ribosómicas 70-kDa/genética , Transducción de Señal/genéticaRESUMEN
Gallbladder cancer is a lethal disease with notable geographical variations worldwide and a predilection towards women. Its main risk factor is prolonged exposure to gallstones, although bacterial infections and other inflammatory conditions are also associated. The recurrent cycles of gallbladder epithelium damage and repair enable a chronic inflammatory environment that promotes progressive morphological impairment through a metaplasia-dysplasia-carcinoma, along with cumulative genome instability. Inactivation of TP53, which is mutated in over 50% of GBC cases, seems to be the earliest and one of the most important carcinogenic pathways involved. Increased cell turnover and oxidative stress promote early alteration of TP53, cell cycle deregulation, apoptosis and replicative senescence. In this review, we will discuss evidence for the role of inflammation in gallbladder carcinogenesis obtained through epidemiological studies, genome-wide association studies, experimental carcinogenesis, morphogenetic studies and comparative studies with other inflammation-driven malignancies. The evidence strongly supports chronic, unresolved inflammation as the main carcinogenic mechanism of gallbladder cancer, regardless of the initial etiologic trigger. Given this central role of inflammation, evaluation of the potential for GBC prevention removing causes of inflammation or using anti-inflammatory drugs in high-risk populations may be warranted.
Asunto(s)
Neoplasias de la Vesícula Biliar/etiología , Inflamación/complicaciones , Enfermedades Autoinmunes/complicaciones , Infecciones Bacterianas/complicaciones , Cálculos Biliares/complicaciones , HumanosRESUMEN
AIMS: Equilibrative nucleoside transporter 1 (ENT1) is the major transporter of the chemotherapeutic drug gemcitabine, the current therapy for advanced gallbladder cancer (GBC). ENT1 expression has been proposed as a predictive marker for gemcitabine-treated pancreatic cancer patients. The aim of study was to explore the value of ENT1 measurement in chemotherapy-naïve patients with advanced GBC. MATERIALS AND RESULTS: Immunohistochemistry for ENT1 was performed on 214 GBC samples from patients who had never undergone co-adjuvant or neo-adjuvant chemotherapy. Advanced GBC cases were divided into groups with low or high ENT1 expression. Kaplan-Meier tests were used for survival analyses. The Cox regression method was used to assess the association of ENT1 expression with overall survival (OS). Low ENT1 expression was associated with younger patient age (P = 0.03) and moderate-to-poor histological differentiation (P = 0.01). pT2 patients with low ENT1 expression had shorter median survival (17.3 versus 28.7 months) and lower OS (17.3% versus 33.3%, P < 0.05) than patients with high ENT1 expression. Low ENT1 expression was an independent prognostic factor for OS (P = 0.036). CONCLUSIONS: ENT1 is a prognostic marker for pT2 GBC patients. Additional studies are needed to determine whether ENT1 has predictive value for gemcitabine response in GBC.
Asunto(s)
Adenocarcinoma/diagnóstico , Antimetabolitos Antineoplásicos/uso terapéutico , Desoxicitidina/análogos & derivados , Tranportador Equilibrativo 1 de Nucleósido/metabolismo , Neoplasias de la Vesícula Biliar/diagnóstico , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/metabolismo , Anciano , Quimioterapia Adyuvante , Desoxicitidina/uso terapéutico , Femenino , Vesícula Biliar/diagnóstico por imagen , Vesícula Biliar/metabolismo , Neoplasias de la Vesícula Biliar/tratamiento farmacológico , Neoplasias de la Vesícula Biliar/metabolismo , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Análisis de Supervivencia , GemcitabinaRESUMEN
BACKGROUND: Gastric cancer (GC) is a deadly malignancy worldwide. In the past, it has been shown that cellular signaling pathway alterations play a crucial role in the development of GC. In particular, deregulation of the PI3K/AKT/mTOR pathway seems to affect multiple GC functions including growth, proliferation, metabolism, motility and angiogenesis. Targeting alterations in this pathway by microRNAs (miRNAs) represents a potential therapeutic strategy, especially in inhibitor-resistant tumors. The objective of this study was to evaluate the expression of 3 pre-selected miRNAs, miR-101-2, miR-125b-2 and miR-451a, in a series of primary GC tissues and matched non-GC tissues and in several GC-derived cell lines, and to subsequently evaluate the functional role of these miRNAs. METHODS: Twenty-five primary GC samples, 25 matched non-GC samples and 3 GC-derived cell lines, i.e., AGS, MKN28 and MKN45, were included in this study. miRNA and target gene expression levels were assessed by quantitative RT-PCR and western blotting, respectively. Subsequently, cell viability, clone formation, cell death, migration and invasion assays were performed on AGS cells. RESULTS: miR-101-2, miR-125b-2 and miR-451a were found to be down-regulated in the primary GC tissues and the GC-derived cell lines tested. MiRNA mimic transfections significantly reduced cell viability and colony formation, increased cell death and reduced cell migration and invasion in AGS cells. We also found that exogenous expression of miR-101-2, miR-125b-2 and miR-451a decreased the expression of their putative targets MTOR, PIK3CB and TSC1, respectively. CONCLUSIONS: Our expression analyses and in vitro functional assays suggest that miR-101-2, miR-125b-2 and miR-451a act as potential tumor suppressors in primary GCs as well as in GC-derived AGS cells.