RESUMEN
Oncogenic alterations to DNA are not transforming in all cellular contexts1,2. This may be due to pre-existing transcriptional programmes in the cell of origin. Here we define anatomic position as a major determinant of why cells respond to specific oncogenes. Cutaneous melanoma arises throughout the body, whereas the acral subtype arises on the palms of the hands, soles of the feet or under the nails3. We sequenced the DNA of cutaneous and acral melanomas from a large cohort of human patients and found a specific enrichment for BRAF mutations in cutaneous melanoma and enrichment for CRKL amplifications in acral melanoma. We modelled these changes in transgenic zebrafish models and found that CRKL-driven tumours formed predominantly in the fins of the fish. The fins are the evolutionary precursors to tetrapod limbs, indicating that melanocytes in these acral locations may be uniquely susceptible to CRKL. RNA profiling of these fin and limb melanocytes, when compared with body melanocytes, revealed a positional identity gene programme typified by posterior HOX13 genes. This positional gene programme synergized with CRKL to amplify insulin-like growth factor (IGF) signalling and drive tumours at acral sites. Abrogation of this CRKL-driven programme eliminated the anatomic specificity of acral melanoma. These data suggest that the anatomic position of the cell of origin endows it with a unique transcriptional state that makes it susceptible to only certain oncogenic insults.
Asunto(s)
Melanoma , Neoplasias Cutáneas , Animales , Animales Modificados Genéticamente , Carcinogénesis/genética , Pie , Mano , Humanos , Melanoma/patología , Uñas , Oncogenes/genética , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Transcripción Genética , Pez Cebra/genética , Melanoma Cutáneo MalignoRESUMEN
Melanomas can have multiple coexisting cell states, including proliferative (PRO) versus invasive (INV) subpopulations that represent a "go or grow" trade-off; however, how these populations interact is poorly understood. Using a combination of zebrafish modeling and analysis of patient samples, we show that INV and PRO cells form spatially structured heterotypic clusters and cooperate in the seeding of metastasis, maintaining cell state heterogeneity. INV cells adhere tightly to each other and form clusters with a rim of PRO cells. Intravital imaging demonstrated cooperation in which INV cells facilitate dissemination of less metastatic PRO cells. We identified the TFAP2 neural crest transcription factor as a master regulator of clustering and PRO/INV states. Isolation of clusters from patients with metastatic melanoma revealed a subset with heterotypic PRO-INV clusters. Our data suggest a framework for the co-existence of these two divergent cell populations, in which heterotypic clusters promote metastasis via cell-cell cooperation.
Asunto(s)
Análisis por Conglomerados , Melanoma/metabolismo , Metástasis de la Neoplasia/patología , Células Neoplásicas Circulantes/patología , Animales , Regulación Neoplásica de la Expresión Génica/fisiología , Melanoma/patología , Cresta Neural/patología , Pez CebraRESUMEN
Oncogenes only transform cells under certain cellular contexts, a phenomenon called oncogenic competence. Using a combination of a human pluripotent stem cellderived cancer model along with zebrafish transgenesis, we demonstrate that the transforming ability of BRAFV600E along with additional mutations depends on the intrinsic transcriptional program present in the cell of origin. In both systems, melanocytes are less responsive to mutations, whereas both neural crest and melanoblast populations are readily transformed. Profiling reveals that progenitors have higher expression of chromatin-modifying enzymes such as ATAD2, a melanoma competence factor that forms a complex with SOX10 and allows for expression of downstream oncogenic and neural crest programs. These data suggest that oncogenic competence is mediated by regulation of developmental chromatin factors, which then allow for proper response to those oncogenes.
Asunto(s)
Carcinogénesis/genética , Carcinogénesis/patología , Cromatina/metabolismo , Melanoma/genética , Melanoma/patología , Cresta Neural/patología , ATPasas Asociadas con Actividades Celulares Diversas/genética , ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Animales , Animales Modificados Genéticamente , Cromatina/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Melanocitos/metabolismo , Melanocitos/patología , Ratones , Neoplasias Experimentales , Células Madre Neoplásicas/patología , Cresta Neural/metabolismo , Células Madre Pluripotentes/patología , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Factores de Transcripción SOXE/genética , Factores de Transcripción SOXE/metabolismo , Transcripción Genética , Pez CebraRESUMEN
Transcriptional profiling of tumors has revealed a stress-like state among the cancer cells with the concerted expression of genes such as fos, jun, and heat-shock proteins, though this has been controversial given possible dissociation-effects associated with single-cell RNA sequencing. Here, we validate the existence of this state using a combination of zebrafish melanoma modeling, spatial transcriptomics, and human samples. We found that the stress-like subpopulation of cancer cells is present from the early stages of tumorigenesis. Comparing with previously reported single-cell RNA sequencing datasets from diverse cancer types, including triple-negative breast cancer, oligodendroglioma, and pancreatic adenocarcinoma, indicated the conservation of this state during tumorigenesis. We also provide evidence that this state has higher tumor-seeding capabilities and that its induction leads to increased growth under both MEK and BRAF inhibitors. Collectively, our study supports the stress-like cells as a cancer cell state expressing a coherent set of genes and exhibiting drug-resistance properties.
Asunto(s)
Carcinogénesis/patología , Melanoma/genética , Estrés Fisiológico/genética , Adenocarcinoma/genética , Animales , Carcinogénesis/metabolismo , Línea Celular Tumoral , Resistencia a Antineoplásicos/genética , Expresión Génica/genética , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Melanoma/metabolismo , Melanoma/fisiopatología , Inhibidores de Proteínas Quinasas/farmacología , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Transcriptoma/genética , Pez CebraRESUMEN
The DNA polymerase Polκ plays a key role in translesion synthesis, an error-prone replication mechanism. Polκ is overexpressed in various tumor types. Here, we found that melanoma and lung and breast cancer cells experiencing stress from oncogene inhibition up-regulated the expression of Polκ and shifted its localization from the cytoplasm to the nucleus. This effect was phenocopied by inhibition of the kinase mTOR, by induction of ER stress, or by glucose deprivation. In unstressed cells, Polκ is continually transported out of the nucleus by exportin-1. Inhibiting exportin-1 or overexpressing Polκ increased the abundance of nuclear-localized Polκ, particularly in response to the BRAFV600E-targeted inhibitor vemurafenib, which decreased the cytotoxicity of the drug in BRAFV600E melanoma cells. These observations were analogous to how Escherichia coli encountering cell stress and nutrient deprivation can up-regulate and activate DinB/pol IV, the bacterial ortholog of Polκ, to induce mutagenesis that enables stress tolerance or escape. However, we found that the increased expression of Polκ was not excessively mutagenic, indicating that noncatalytic or other functions of Polκ could mediate its role in stress responses in mammalian cells. Repressing the expression or nuclear localization of Polκ might prevent drug resistance in some cancer cells.
Asunto(s)
ADN Polimerasa Dirigida por ADN/metabolismo , Resistencia a Antineoplásicos , Melanoma/enzimología , Proteínas de Neoplasias/metabolismo , Transducción de Señal , Línea Celular Tumoral , ADN Polimerasa Dirigida por ADN/genética , Humanos , Melanoma/tratamiento farmacológico , Melanoma/genética , Proteínas de Neoplasias/genéticaRESUMEN
Developmental processes underlying normal tissue regeneration have been implicated in cancer, but the degree of their enactment during tumor progression and under the selective pressures of immune surveillance, remain unknown. Here we show that human primary lung adenocarcinomas are characterized by the emergence of regenerative cell types, typically seen in response to lung injury, and by striking infidelity among transcription factors specifying most alveolar and bronchial epithelial lineages. In contrast, metastases are enriched for key endoderm and lung-specifying transcription factors, SOX2 and SOX9, and recapitulate more primitive transcriptional programs spanning stem-like to regenerative pulmonary epithelial progenitor states. This developmental continuum mirrors the progressive stages of spontaneous outbreak from metastatic dormancy in a mouse model and exhibits SOX9-dependent resistance to natural killer cells. Loss of developmental stage-specific constraint in macrometastases triggered by natural killer cell depletion suggests a dynamic interplay between developmental plasticity and immune-mediated pruning during metastasis.
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Adenocarcinoma/inmunología , Adenocarcinoma/patología , Sistema Inmunológico/fisiología , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/patología , Metástasis de la Neoplasia , Animales , Bronquios/metabolismo , Diferenciación Celular , Linaje de la Célula , Análisis por Conglomerados , Bases de Datos Genéticas , Progresión de la Enfermedad , Endodermo/metabolismo , Femenino , Humanos , Hidrogeles/química , Células Asesinas Naturales/metabolismo , Pulmón/patología , Ratones , Fenotipo , Alveolos Pulmonares/metabolismo , Regeneración , Transducción de SeñalRESUMEN
Transgenic animals are invaluable for modeling cancer genomics, but often require complex crosses of multiple germline alleles to obtain the desired combinations. Zebrafish models have advantages in that transgenes can be rapidly tested by mosaic expression, but typically lack spatial and temporal control of tumor onset, which limits their utility for the study of tumor progression and metastasis. To overcome these limitations, we have developed a method referred to as Transgene Electroporation in Adult Zebrafish (TEAZ). TEAZ can deliver DNA constructs with promoter elements of interest to drive fluorophores, oncogenes or CRISPR-Cas9-based mutagenic cassettes in specific cell types. Using TEAZ, we created a highly aggressive melanoma model via Cas9-mediated inactivation of Rb1 in the context of BRAFV600E in spatially constrained melanocytes. Unlike prior models that take â¼4â months to develop, we found that TEAZ leads to tumor onset in â¼7â weeks, and these tumors develop in fully immunocompetent animals. As the resulting tumors initiated at highly defined locations, we could track their progression via fluorescence, and documented deep invasion into tissues and metastatic deposits. TEAZ can be deployed to other tissues and cell types, such as the heart, with the use of suitable transgenic promoters. The versatility of TEAZ makes it widely accessible for rapid modeling of somatic gene alterations and cancer progression at a scale not achievable in other in vivo systems.
Asunto(s)
Envejecimiento/genética , Electroporación , Transgenes , Pez Cebra/genética , Animales , Animales Modificados Genéticamente , Sistemas CRISPR-Cas/genética , Carcinogénesis/genética , Carcinogénesis/patología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Embrión no Mamífero/metabolismo , Técnicas de Transferencia de Gen , Melanoma/patología , Plásmidos/genética , Regiones Promotoras Genéticas , Pez Cebra/embriologíaRESUMEN
Advanced, metastatic melanomas frequently grow in subcutaneous tissues and portend a poor prognosis. Though subcutaneous tissues are largely composed of adipocytes, the mechanisms by which adipocytes influence melanoma are poorly understood. Using in vitro and in vivo models, we find that adipocytes increase proliferation and invasion of adjacent melanoma cells. Additionally, adipocytes directly transfer lipids to melanoma cells, which alters tumor cell metabolism. Adipocyte-derived lipids are transferred to melanoma cells through the FATP/SLC27A family of lipid transporters expressed on the tumor cell surface. Among the six FATP/SLC27A family members, melanomas significantly overexpress FATP1/SLC27A1. Melanocyte-specific FATP1 expression cooperates with BRAFV600E in transgenic zebrafish to accelerate melanoma development, an effect that is similarly seen in mouse xenograft studies. Pharmacologic blockade of FATPs with the small-molecule inhibitor Lipofermata abrogates lipid transport into melanoma cells and reduces melanoma growth and invasion. These data demonstrate that stromal adipocytes can drive melanoma progression through FATP lipid transporters and represent a new target aimed at interrupting adipocyte-melanoma cross-talk.Significance: We demonstrate that stromal adipocytes are donors of lipids that mediate melanoma progression. Adipocyte-derived lipids are taken up by FATP proteins that are aberrantly expressed in melanoma. Inhibition of FATPs decreases melanoma lipid uptake, invasion, and growth. We provide a mechanism for how stromal adipocytes drive tumor progression and demonstrate a novel microenvironmental therapeutic target. Cancer Discov; 8(8); 1006-25. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 899.
Asunto(s)
Adipocitos/citología , Proteínas de Transporte de Ácidos Grasos/metabolismo , Ácidos Grasos/metabolismo , Melanoma/patología , Proteínas Proto-Oncogénicas B-raf/genética , Células 3T3-L1 , Adipocitos/metabolismo , Animales , Animales Modificados Genéticamente , Transporte Biológico/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Técnicas de Cocultivo , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica , Humanos , Melanoma/tratamiento farmacológico , Melanoma/metabolismo , Ratones , Mutación , Invasividad Neoplásica , Trasplante de Neoplasias , Compuestos de Espiro/administración & dosificación , Compuestos de Espiro/farmacología , Tiadiazoles/administración & dosificación , Tiadiazoles/farmacología , Microambiente Tumoral , Regulación hacia Arriba , Pez CebraRESUMEN
Patterning of vertebrate melanophores is essential for mate selection and protection from UV-induced damage. Patterning can be influenced by circulating long-range factors, such as hormones, but it is unclear how their activity is controlled in recipient cells to prevent excesses in cell number and migration. The zebrafish wanderlust mutant harbors a mutation in the sheddase bace2 and exhibits hyperdendritic and hyperproliferative melanophores that localize to aberrant sites. We performed a chemical screen to identify suppressors of the wanderlust phenotype and found that inhibition of insulin/PI3Kγ/mTOR signaling rescues the defect. In normal physiology, Bace2 cleaves the insulin receptor, whereas its loss results in hyperactive insulin/PI3K/mTOR signaling. Insulin B, an isoform enriched in the head, drives the melanophore defect. These results suggest that insulin signaling is negatively regulated by melanophore-specific expression of a sheddase, highlighting how long-distance factors can be regulated in a cell-type-specific manner.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/metabolismo , Tipificación del Cuerpo , Insulina/metabolismo , Melanóforos/fisiología , Pigmentación , Proteínas de Pez Cebra/metabolismo , Pez Cebra/fisiología , Secretasas de la Proteína Precursora del Amiloide/genética , Animales , Movimiento Celular/fisiología , Embrión no Mamífero/citología , Embrión no Mamífero/fisiología , Regulación del Desarrollo de la Expresión Génica , Insulina/genética , Melanóforos/citología , Mutación , Fenotipo , Fosfatidilinositol 3-Quinasas , Transducción de Señal , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Pez Cebra/embriología , Proteínas de Pez Cebra/genéticaRESUMEN
Transcriptional deregulation of oncogenic pathways is a hallmark of cancer and can be due to epigenetic alterations. 5-Hydroxymethylcytosine (5-hmC) is an epigenetic modification that has not been studied in pancreatic cancer. Genome-wide analysis of 5-hmC-enriched loci with hmC-seal was conducted in a cohort of low-passage pancreatic cancer cell lines, primary patient-derived xenografts, and pancreatic controls and revealed strikingly altered patterns in neoplastic tissues. Differentially hydroxymethylated regions preferentially affected known regulatory regions of the genome, specifically overlapping with known H3K4me1 enhancers. Furthermore, base pair resolution analysis of cytosine methylation and hydroxymethylation with oxidative bisulfite sequencing was conducted and correlated with chromatin accessibility by ATAC-seq and gene expression by RNA-seq in pancreatic cancer and control samples. 5-hmC was specifically enriched at open regions of chromatin, and gain of 5-hmC was correlated with up-regulation of the cognate transcripts, including many oncogenic pathways implicated in pancreatic neoplasia, such as MYC, KRAS, VEGFA, and BRD4 Specifically, BRD4 was overexpressed and acquired 5-hmC at enhancer regions in the majority of neoplastic samples. Functionally, acquisition of 5-hmC at BRD4 promoter was associated with increase in transcript expression in reporter assays and primary samples. Furthermore, blockade of BRD4 inhibited pancreatic cancer growth in vivo. In summary, redistribution of 5-hmC and preferential enrichment at oncogenic enhancers is a novel regulatory mechanism in human pancreatic cancer.
Asunto(s)
5-Metilcitosina/análogos & derivados , Neoplasias Pancreáticas/genética , Secuencias Reguladoras de Ácidos Nucleicos , Análisis de Secuencia de ARN/métodos , 5-Metilcitosina/metabolismo , Animales , Línea Celular Tumoral , Epigénesis Genética , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Estudio de Asociación del Genoma Completo , Histonas/metabolismo , Humanos , Ratones , Trasplante de Neoplasias , Modelación Específica para el PacienteRESUMEN
Metastasis is the defining feature of advanced malignancy, yet remains challenging to study in laboratory environments. Here, we describe a high-throughput zebrafish system for comprehensive, in vivo assessment of metastatic biology. First, we generated several stable cell lines from melanomas of transgenic mitfa-BRAF(V600E);p53(-/-) fish. We then transplanted the melanoma cells into the transparent casper strain to enable highly quantitative measurement of the metastatic process at single-cell resolution. Using computational image analysis of the resulting metastases, we generated a metastasis score, µ, that can be applied to quantitative comparison of metastatic capacity between experimental conditions. Furthermore, image analysis also provided estimates of the frequency of metastasis-initiating cells (â¼1/120,000 cells). Finally, we determined that the degree of pigmentation is a key feature defining cells with metastatic capability. The small size and rapid generation of progeny combined with superior imaging tools make zebrafish ideal for unbiased high-throughput investigations of cell-intrinsic or microenvironmental modifiers of metastasis. The approaches described here are readily applicable to other tumor types and thus serve to complement studies also employing murine and human cell culture systems.
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Modelos Biológicos , Neoplasias/patología , Pez Cebra , Algoritmos , Animales , Animales Modificados Genéticamente , Línea Celular Tumoral , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Melanoma/genética , Melanoma/metabolismo , Melanoma/patología , Mutación , Metástasis de la Neoplasia , Neoplasias/genética , Neoplasias/metabolismo , TranscriptomaRESUMEN
MicroRNAs (miRNAs) are small non-coding RNAs, which inhibit the stability and/or translation of a mRNA. miRNAs have been found to play a powerful role in various cardiovascular diseases. Recently, we have demonstrated that a microRNA (miR-181c) can be encoded in the nucleus, processed to the mature form in the cytosol, translocated into the mitochondria, and ultimately can regulate mitochondrial gene expression. However the in vivo impact of miR-181c is unknown. Here we report an in-vivo method for administration of miR-181c in rats, which leads to reduced exercise capacity and signs of heart failure, by targeting the 3'-end of mt-COX1 (cytochrome c oxidase subunit 1). We cloned miR-181c and packaged it in lipid-based nanoparticles for systemic delivery. The plasmid DNA complexed nanovector shows no apparent toxicity. We find that the mRNA levels of mitochondrial complex IV genes in the heart, but not any other mitochondrial genes, are significantly altered with miR-181c overexpression, suggesting selective mitochondrial complex IV remodeling due to miR-181c targeting mt-COX1. Isolated heart mitochondrial studies showed significantly altered O2-consumption, ROS production, matrix calcium, and mitochondrial membrane potential in miR-181c-treated animals. For the first time, this study shows that miRNA delivered to the heart in-vivo can lead to cardiac dysfunction by regulating mitochondrial genes.
Asunto(s)
Metabolismo Energético/genética , Genes Mitocondriales/genética , Predisposición Genética a la Enfermedad/genética , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/metabolismo , MicroARNs/genética , Animales , ADN/administración & dosificación , ADN/química , ADN/genética , Liposomas , Masculino , Nanopartículas/química , Ratas , Ratas Sprague-DawleyRESUMEN
5-hydroxymethylcytosine (5-hmC) is a recently discovered epigenetic modification that is altered in cancers. Genome-wide assays for 5-hmC determination are needed as many of the techniques for 5-methylcytosine (5-mC) determination, including methyl-sensitive restriction digestion and bisulfite sequencing cannot distinguish between 5-mC and 5-hmC. Glycosylation of 5-hmC residues by beta-glucosyl transferase (ß-GT) can make CCGG residues insensitive to digestion by MspI. Restriction digestion by HpaII, MspI or MspI after ß-GT conversion, followed by adapter ligation, massive parallel sequencing and custom bioinformatic analysis allowed us determine distribution of 5-mC and 5-hmC at single base pair resolution at MspI restriction sites. The resulting HpaII tiny fragment Enrichment by Ligation-mediated PCR with ß-GT (HELP-GT) assay identified 5-hmC loci that were validated at global level by liquid chromatography-mass spectrometry (LC-MS) and the locus-specific level by quantitative reverse transcriptase polymerase chain reaction of 5-hmC pull-down DNA. Hydroxymethylation at both promoter and intragenic locations correlated positively with gene expression. Analysis of pancreatic cancer samples revealed striking redistribution of 5-hmC sites in cancer cells and demonstrated enrichment of this modification at many oncogenic promoters such as GATA6. The HELP-GT assay allowed global determination of 5-hmC and 5-mC from low amounts of DNA and with the use of modest sequencing resources. Redistribution of 5-hmC seen in cancer highlights the importance of determination of this modification in conjugation with conventional methylome analysis.
Asunto(s)
Citosina/análogos & derivados , ADN de Neoplasias/química , 5-Metilcitosina/análisis , Animales , Línea Celular Tumoral , Transformación Celular Neoplásica , Citosina/análisis , Citosina/metabolismo , Expresión Génica , Genoma Humano , Genómica/métodos , Glicosiltransferasas/metabolismo , Humanos , Ratones , Neoplasias Pancreáticas/genética , Reacción en Cadena de la PolimerasaRESUMEN
PURPOSE: Recent microarray and RNA-sequencing studies have uncovered aberrantly expressed microRNAs (miRNA) in Barrett's esophagus-associated esophageal adenocarcinoma. The functional significance of these miRNAs in esophageal adenocarcinoma initiation and progression is largely unknown. EXPERIMENTAL DESIGN: Expression levels of miR-199a/b-3p, -199a-5p, -199b-5p, -200b, -200c, -223, and -375 were determined in microdissected tissues from cardiac mucosa, Barrett's esophagus, dysplastic Barrett's esophagus, and esophageal adenocarcinoma using quantitative real-time PCR. miR-223 expression was validated in precursors and esophageal adenocarcinomas from 95 patients with esophageal adenocarcinoma by in situ hybridization (ISH). miR-223 was transfected into two esophageal adenocarcinoma cell lines, and in vitro assays were conducted. Target genes were identified using Illumina microarray, and results were validated in cell lines and human specimens. RESULTS: miR-199 family members and miR-223 were significantly overexpressed in esophageal adenocarcinoma, however, only miR-223 showed a stepwise increase during esophageal adenocarcinoma carcinogenesis. A similar trend was observed by ISH, which additionally showed that miR-223 is exclusively expressed by the epithelial compartment. miR-223-overexpressing cells had statistically significantly more migratory and invasive potential than scramble sequence-transfected cells. PARP1 was identified as a direct target gene of miR-223 in esophageal adenocarcinoma cells. Increased sensitivity to chemotherapy was observed in cells with enforced miR-223 expression and reduced PARP1. CONCLUSIONS: miR-223 is significantly upregulated during the Barrett's esophagus-dysplasia-esophageal adenocarcinoma sequence. Although high miR-223 levels might contribute to an aggressive phenotype, our results also suggest that patients with esophageal adenocarcinoma with high miR-223 levels might benefit from treatment with DNA-damaging agents.
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Esófago de Barrett/tratamiento farmacológico , Neoplasias Esofágicas/tratamiento farmacológico , MicroARNs/biosíntesis , Poli(ADP-Ribosa) Polimerasas/biosíntesis , Adenocarcinoma/genética , Adenocarcinoma/patología , Esófago de Barrett/genética , Esófago de Barrett/patología , Progresión de la Enfermedad , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patología , Regulación Neoplásica de la Expresión Génica , Humanos , Hibridación in Situ , Poli(ADP-Ribosa) Polimerasa-1 , Regulación hacia ArribaRESUMEN
Pancreatic ductal adenocarcinoma (PDA) remains a lethal human malignancy with historically limited success in treatment. The role of aberrant Notch signaling, which requires the constitutive activation of γ-secretase, in the initiation and progression of PDA is well defined and inhibitors of this pathway are currently in clinical trials. Here we investigated the in vivo therapeutic effect of PF-03084014, a selective γ-secretase inhibitor, alone and in combination with gemcitabine in pancreatic cancer xenografts. PF-03084014 treatment inhibited the cleavage of nuclear Notch 1 intracellular domain and Notch targets Hes-1 and Hey-1. Gemcitabine treatment showed good response but not capable of inducing tumor regressions and targeting the tumor-resident cancer stem cells (CD24(+)CD44(+) and ALDH(+) tumor cells). A combination of PF-03084014 and gemcitabine treatment resulted tumor regression in 3 of 4 subcutaneously implanted xenograft models. PF-03084014, and in combination with gemcitabine reduced putative cancer stem cells, indicating that PF-03084014 target the especially dangerous and resilient cancer stem cells within pancreatic tumors. Tumor re-growth curves plotted after drug treatments demonstrated that the effect of the combination therapy was sustainable than that of gemcitabine. Notably, in a highly aggressive orthotopic model, PF-03084014 and gemcitabine combination was effective in inducing apoptosis, inhibition of tumor cell proliferation and angiogenesis, resulting in the attenuation of primary tumor growth as well as controlling metastatic dissemination, compared to gemcitabine treatment. In summary, our preclinical data suggest that PF-03084014 has greater anti-tumor activity in combination with gemcitabine in PDA and provides rationale for further investigation of this combination in PDA.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Carcinoma Ductal Pancreático/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pancreáticas/metabolismo , Receptores Notch/metabolismo , Animales , Antígenos CD/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Apoptosis/efectos de los fármacos , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/secundario , Proliferación Celular/efectos de los fármacos , Desoxicitidina/administración & dosificación , Desoxicitidina/análogos & derivados , Progresión de la Enfermedad , Femenino , Humanos , Neoplasias Hepáticas/prevención & control , Neoplasias Hepáticas/secundario , Neoplasias Pulmonares/prevención & control , Neoplasias Pulmonares/secundario , Ratones , Ratones Desnudos , Terapia Molecular Dirigida , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/patología , Receptores Notch/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Tetrahidronaftalenos/administración & dosificación , Carga Tumoral/efectos de los fármacos , Células Tumorales Cultivadas , Valina/administración & dosificación , Valina/análogos & derivados , Ensayos Antitumor por Modelo de Xenoinjerto , GemcitabinaRESUMEN
CONTEXT: Although pancreatic cancer is a common, highly lethal malignancy, the molecular events that enable precursor lesions to become invasive carcinoma remain unclear. We previously reported that the high-mobility group A1 (HMGA1) protein is overexpressed in >90% of primary pancreatic cancers, with absent or low levels in early precursor lesions. METHODS: Here, we investigate the role of HMGA1 in reprogramming pancreatic epithelium into invasive cancer cells. We assessed oncogenic properties induced by HMGA1 in non-transformed pancreatic epithelial cells expressing activated K-RAS. We also explored the HMGA1-cyclooxygenase (COX-2) pathway in human pancreatic cancer cells and the therapeutic effects of COX-2 inhibitors in xenograft tumorigenesis. RESULTS: HMGA1 cooperates with activated K-RAS to induce migration, invasion, and anchorage-independent cell growth in a cell line derived from normal human pancreatic epithelium. Moreover, HMGA1 and COX-2 expression are positively correlated in pancreatic cancer cell lines (r(2) = 0.93; p < 0.001). HMGA1 binds directly to the COX-2 promoter at an AT-rich region in vivo in three pancreatic cancer cell lines. In addition, HMGA1 induces COX-2 expression in pancreatic epithelial cells, while knock-down of HMGA1 results in repression of COX-2 in pancreatic cancer cells. Strikingly, we also discovered that Sulindac (a COX-1/COX-2 inhibitor) or Celecoxib (a more specific COX-2 inhibitor) block xenograft tumorigenesis from pancreatic cancer cells expressing high levels of HMGA1. CONCLUSIONS: Our studies identify for the first time an important role for the HMGA1-COX-2 pathway in pancreatic cancer and suggest that targeting this pathway could be effective to treat, or even prevent, pancreatic cancer.
Asunto(s)
Adenocarcinoma/genética , Ciclooxigenasa 2/genética , Proteína HMGA1a/genética , Neoplasias Pancreáticas/genética , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/prevención & control , Animales , Celecoxib , División Celular/genética , Línea Celular Tumoral , Movimiento Celular/genética , Ciclooxigenasa 2/fisiología , Inhibidores de la Ciclooxigenasa/administración & dosificación , Expresión Génica , Proteína HMGA1a/fisiología , Humanos , Ratones , Ratones Desnudos , Invasividad Neoplásica/genética , Metástasis de la Neoplasia/genética , Trasplante de Neoplasias , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/prevención & control , Pirazoles/administración & dosificación , Sulfonamidas/administración & dosificación , Sulindac/administración & dosificación , Trasplante Heterólogo , Proteínas ras/fisiologíaRESUMEN
MicroRNA (miRs) have emerged as salient regulators in cancer homeostasis and, recently, as putative therapeutics. Cholangiocarcinomas (CCA) are aggressive cancers with survival usually measured in months. mRNA arrays followed by pathway analysis revealed that miR-494 is a major modulator of the cell cycle progression from gap 2 (G2) to mitosis (M). We performed fluorescence activated cell sorting (FACS) as well as differential interference contrast (DIC) microscopy, and confirmed that miR-494 induces a significant arrest in G2/M in CCA cells. Furthermore, we verified that miR-494 modulates the protein level of six genes involved in the G2/M transition: Polo-like Kinase 1 (PLK1), pituitary tumor-transforming gene 1 (PTTG1), Cyclin B1 (CCNB1), cell-division cycle 2 (CDC2), cell-division cycle 20 (CDC20) and topoisomerase II α (TOP2A). Next, we identified direct binding of miR-494 to the open reading frame (ORF) and downregulation of PTTG1 and TOP2A. In summary, our findings suggest that miR-494 has a global regulatory role in cell cycle progression, exerted by concerted effects on multiple proteins involved in gap 1 (G1) to synthesis (S), as described previously, as well as G2 to M progression. Therefore, it appears that the simultaneous effects of a single miR species on multiple targets along the same canonical pathway is advantageous for the usage of miRs as therapeutics. In addition, our data suggest that miRs act within a narrow range. miR expression above the upper threshold does not appear to induce further effects, which is reassuring in terms of off-target effects of miR surrounding noncancerous tissue.
Asunto(s)
MicroARNs/metabolismo , Antígenos de Neoplasias/metabolismo , Secuencia de Bases , Sitios de Unión , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Colangiocarcinoma/metabolismo , Colangiocarcinoma/patología , Ciclina B1/metabolismo , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo , Citometría de Flujo , Puntos de Control de la Fase G2 del Ciclo Celular , Humanos , Puntos de Control de la Fase M del Ciclo Celular , Proteínas de Neoplasias/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Securina , Regiones no Traducidas , Quinasa Tipo Polo 1RESUMEN
Acquired chemotherapy resistance is a major contributor to treatment failure in oncology. For example, the efficacy of the common anticancer agent doxorubicin (DOX) is limited by the emergence of multidrug resistance (MDR) phenotype in cancer cells. While dose escalation of DOX can circumvent such resistance to a degree, this is precluded by the appearance of cardiotoxicity, a particularly debilitating condition in children. In vitro studies have established the ability of the natural phytochemical curcumin to overcome MDR; however, its widespread clinical application is restricted by poor solubility and low bioavailability. Building upon our recently developed polymer nanoparticle of curcumin (NanoCurc or NC) that significantly enhances the systemic bioavailability of curcumin, we synthesized a doxorubicin-curcumin composite nanoparticle formulation called NanoDoxCurc (NDC) for overcoming DOX resistance. Compared to DOX alone, NDC inhibited the MDR phenotype and caused striking growth inhibition both in vitro and in vivo in several models of DOX-resistant cancers (multiple myeloma, acute leukemia, prostate and ovarian cancers, respectively). Notably, NDC-treated mice also demonstrated complete absence of cardiac toxicity, as assessed by echocardiography, or any bone marrow suppression, even at cumulative dosages where free DOX and pegylated liposomal DOX (Doxil®) resulted in demonstrable attenuation of cardiac function and hematological toxicities. This improvement in safety profile was achieved through a reduction of DOX-induced intracellular oxidative stress, as indicated by total glutathione levels and glutathione peroxidase activity in cardiac tissue. A composite DOX-curcumin nanoparticle that overcomes both MDR-based DOX chemoresistance and DOX-induced cardiotoxicity holds promise for providing lasting and safe anticancer therapy.
Asunto(s)
Antibióticos Antineoplásicos/administración & dosificación , Cardiomiopatías/prevención & control , Doxorrubicina/análogos & derivados , Doxorrubicina/administración & dosificación , Nanopartículas/administración & dosificación , Animales , Antibióticos Antineoplásicos/efectos adversos , Antibióticos Antineoplásicos/química , Cardiomiopatías/inducido químicamente , Cardiomiopatías/tratamiento farmacológico , Línea Celular Tumoral , Curcumina/administración & dosificación , Curcumina/análogos & derivados , Curcumina/química , Doxorrubicina/efectos adversos , Doxorrubicina/química , Resistencia a Antineoplásicos , Glutatión/metabolismo , Humanos , Masculino , Ratones , Ratones Desnudos , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/metabolismo , Nanopartículas/química , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/metabolismo , Distribución Aleatoria , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy, with most patients facing an adverse clinical outcome. Aberrant Notch pathway activation has been implicated in the initiation and progression of PDAC, specifically the aggressive phenotype of the disease. We used a panel of human PDAC cell lines as well as patient-derived PDAC xenografts to determine whether pharmacologic targeting of Notch pathway could inhibit PDAC growth and potentiate gemcitabine sensitivity. MRK-003, a potent and selective γ-secretase inhibitor, treatment resulted in the downregulation of nuclear Notch1 intracellular domain, inhibition of anchorage-independent growth, and reduction of tumor-initiating cells capable of extensive self-renewal. Pretreatment of PDAC cells with MRK-003 in cell culture significantly inhibited the subsequent engraftment in immunocompromised mice. MRK-003 monotherapy significantly blocked tumor growth in 5 of 9 (56%) PDAC xenografts. A combination of MRK-003 and gemcitabine showed enhanced antitumor effects compared with gemcitabine in 4 of 9 (44%) PDAC xenografts, reduced tumor cell proliferation, and induced both apoptosis and intratumoral necrosis. Gene expression analysis of untreated tumors indicated that upregulation of NF-κB pathway components was predictive of sensitivity to MRK-003, whereas upregulation in B-cell receptor signaling and nuclear factor erythroid-derived 2-like 2 pathway correlated with response to the combination of MRK-003 with gemcitabine. Our findings strengthen the rationale for small-molecule inhibition of Notch signaling as a therapeutic strategy in PDAC.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Antineoplásicos/farmacología , Óxidos S-Cíclicos/farmacología , Neoplasias Pancreáticas/tratamiento farmacológico , Tiadiazoles/farmacología , Animales , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Adhesión Celular , Línea Celular Tumoral , Núcleo Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Óxidos S-Cíclicos/uso terapéutico , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Desoxicitidina/uso terapéutico , Sinergismo Farmacológico , Expresión Génica/efectos de los fármacos , Humanos , Masculino , Ratones , Ratones Desnudos , Células Madre Neoplásicas/efectos de los fármacos , Análisis de Secuencia por Matrices de Oligonucleótidos , Neoplasias Pancreáticas/patología , Receptor Notch1/antagonistas & inhibidores , Receptor Notch1/genética , Receptor Notch1/metabolismo , Tiadiazoles/uso terapéutico , Transcriptoma/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , GemcitabinaRESUMEN
Mucin 16 (cancer antigen 125) is a cell surface glycoprotein that plays a role in promoting cancer cell growth in ovarian cancer. The aims of this study were to examine mucin 16 expression in a large number of digestive tract adenocarcinomas and precursors and to determine whether mucin 16 up-regulation is correlated with patient outcome. Tissue microarrays were constructed using surgical resection tissues and included pancreatic (115 normal, 29 precursors, 200 pancreatic ductal adenocarcinomas), esophageal (86 normal, 104 precursors, 95 esophageal adenocarcinomas, 35 lymph node metastases), gastric (211 normal, 8 precursors, 119 gastric adenocarcinomas, 62 lymph node metastases), and colorectal (34 normal, 17 precursors, 39 colorectal adenocarcinomas) tissues. Mucin 16 was detected in 81.5%, 69.9%, 41.2%, and 64.1% of the pancreatic ductal adenocarcinomas, esophageal adenocarcinomas, gastric adenocarcinomas, and colorectal adenocarcinomas, respectively. Mucin 16 was seen in a subset of the precursors. On multivariate analysis, moderate/diffuse mucin 16 in pancreatic ductal adenocarcinomas was strongly associated with poor survival (P < .001), independent of other prognosis predictors. A similar trend was observed for esophageal adenocarcinomas (P = .160) and gastric adenocarcinomas (P = .080). Focal mucin 16 in colorectal adenocarcinomas was significantly correlated (P = .044) with a better patient outcome, when compared with mucin 16-negative cases. Using Western blot analysis, we found mucin 16 expression in 3 of 6 pancreatic ductal adenocarcinoma and 1 of 2 esophageal adenocarcinoma cell lines. We conclude that most of the digestive tract adenocarcinomas and a subset of their precursors express mucin 16. Mucin 16 expression is an independent predictor of poor outcome in pancreatic ductal adenocarcinomas and potentially in esophageal adenocarcinomas and gastric adenocarcinomas. We propose that mucin 16 may function as a prognostic marker and therapeutic target in the future.