RESUMEN
Altered energy metabolism is a cancer hallmark as malignant cells tailor their metabolic pathways to meet their energy requirements. Glucose and glutamine are the major nutrients that fuel cellular metabolism, and the pathways utilizing these nutrients are often altered in cancer. Here, we show that the long ncRNA CCAT2, located at the 8q24 amplicon on cancer risk-associated rs6983267 SNP, regulates cancer metabolism in vitro and in vivo in an allele-specific manner by binding the Cleavage Factor I (CFIm) complex with distinct affinities for the two subunits (CFIm25 and CFIm68). The CCAT2 interaction with the CFIm complex fine-tunes the alternative splicing of Glutaminase (GLS) by selecting the poly(A) site in intron 14 of the precursor mRNA. These findings uncover a complex, allele-specific regulatory mechanism of cancer metabolism orchestrated by the two alleles of a long ncRNA.
Asunto(s)
Glutaminasa/genética , Neoplasias/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Factores de Escisión y Poliadenilación de ARNm/metabolismo , Alelos , Empalme Alternativo , Metabolismo Energético , Células HCT116 , Humanos , Neoplasias/genética , Precursores del ARN/química , Precursores del ARN/metabolismo , ARN Mensajero/metabolismoRESUMEN
BACKGROUND: Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype that lacks targeted therapies. Patients with TNBC have a very poor prognosis because the disease often metastasizes. New treatment approaches addressing drivers of metastasis and tumor growth are crucial to improving patient outcomes. Developing targeted gene therapy is thus a high priority for TNBC patients. PEA15 (phosphoprotein enriched in astrocytes, 15 kDa) is known to bind to ERK, preventing ERK from being translocated to the nucleus and hence blocking its activity. The biological function of PEA15 is tightly regulated by its phosphorylation at Ser104 and Ser116. However, the function and impact of phosphorylation status of PEA15 in the regulation of TNBC metastasis and in epithelial-to-mesenchymal transition (EMT) are not well understood. METHODS: We established stable cell lines overexpressing nonphosphorylatable (PEA15-AA) and phospho-mimetic (PEA15-DD) mutants. To dissect specific cellular mechanisms regulated by PEA15 phosphorylation status, we performed RT-PCR immune and metastasis arrays. In vivo mouse models were used to determine the effects of PEA15 phosphorylation on tumor growth and metastasis. RESULTS: We found that the nonphosphorylatable mutant PEA15-AA prevented formation of mammospheres and expression of EMT markers in vitro and decreased tumor growth and lung metastasis in in vivo experiments when compared to control, PEA15-WT and phosphomimetic PEA15-DD. However, phosphomimetic mutant PEA15-DD promoted migration, mesenchymal marker expression, tumorigenesis, and lung metastasis in the mouse model. PEA15-AA-mediated inhibition of breast cancer cell migratory capacity and tumorigenesis was the partial result of decreased expression of interleukin-8 (IL-8). Further, we identified that expression of IL-8 was possibly mediated through one of the ERK downstream molecules, Ets-1. CONCLUSIONS: Our results show that PEA15 phosphorylation status serves as an important regulator for PEA15's dual role as an oncogene or tumor suppressor and support the potential of PEA15-AA as a therapeutic strategy for treatment of TNBC.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/genética , Transición Epitelial-Mesenquimal , Neoplasias de la Mama Triple Negativas , Animales , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Transición Epitelial-Mesenquimal/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Interleucina-8 , Ratones , Neoplasias de la Mama Triple Negativas/genéticaRESUMEN
BACKGROUND: Poor outcomes for patients with ovarian cancer relate to dormant, drug-resistant cancer cells that survive after primary surgery and chemotherapy. Ovarian cancer (OvCa) cells persist in poorly vascularized scars on the peritoneal surface and depend on autophagy to survive nutrient deprivation. The authors have sought drugs that target autophagic cancer cells selectively to eliminate residual disease. METHODS: By using unbiased small-interfering RNA (siRNA) screens, the authors observed that knockdown of anaplastic lymphoma kinase (ALK) reduced the survival of autophagic OvCa cells. Small-molecule ALK inhibitors were evaluated for their selective toxicity against autophagic OvCa cell lines and xenografts. Autophagy was induced by reexpression of GTP-binding protein Di-Ras3 (DIRAS3) or serum starvation and was evaluated with Western blot analysis, fluorescence imaging, and transmission electron microscopy. Signaling pathways required for crizotinib-induced apoptosis of autophagic cells were explored with flow cytometric analysis, Western blot analysis, short-hairpin RNA knockdown of autophagic proteins, and small-molecule inhibitors of STAT3 and BCL-2. RESULTS: Induction of autophagy by reexpression of DIRAS3 or serum starvation in multiple OvCa cell lines significantly reduced the 50% inhibitory concentration of crizotinib and other ALK inhibitors. In 2 human OvCa xenograft models, the DIRAS3-expressing tumors treated with crizotinib had significantly decreased tumor burden and long-term survival in 67% to 79% of mice. Crizotinib treatment of autophagic cancer cells further enhanced autophagy and induced autophagy-mediated apoptosis by decreasing phosphorylated STAT3 and BCL-2 signaling. CONCLUSIONS: Crizotinib may eliminate dormant, autophagic, drug-resistant OvCa cells that remain after conventional cytoreductive surgery and combination chemotherapy. A clinical trial of ALK inhibitors as maintenance therapy after second-look operations should be seriously considered.
Asunto(s)
Quinasa de Linfoma Anaplásico/genética , Neoplasias Ováricas/tratamiento farmacológico , Factor de Transcripción STAT3/genética , Proteínas de Unión al GTP rho/genética , Quinasa de Linfoma Anaplásico/antagonistas & inhibidores , Animales , Autofagia/efectos de los fármacos , Línea Celular Tumoral , Linaje de la Célula/genética , Supervivencia Celular/genética , Crizotinib/farmacología , Resistencia a Antineoplásicos/genética , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Xenoinjertos , Humanos , Ratones , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Inhibidores de Proteínas Quinasas/farmacología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacología , Transducción de Señal/efectos de los fármacosRESUMEN
This report details a search for alternative strains that produce the diterpenoid sphaeropsidin A (SphA) among A. candidus strains from the USDA Northern Regional Research Laboratories Culture Collection. We identified two strains that produced SphA using a limited set of test media. An initial scaled-up fermentation of NRRL 313 and isolation effort led to the procurement of sufficient quantities of SphA to prepare five semi-synthetic analogues (1-5) and evaluate their anticancer effects against glioblastoma cells D423 and Gli56 grown in 2D and 3D cultures. Although, the effectiveness of the synthetic analogues varied depending on the cell line and the type of cell culture, compound 5, bearing an aromatic ring at C16, displayed a stronger toxicity towards both D423 and Gli56 cell lines in 2D cultures and D423 spheroids in 3D culture than either SphA or compounds 1-4.
Asunto(s)
Antineoplásicos Fitogénicos/síntesis química , Aspergillus/química , Diterpenos/química , Antineoplásicos Fitogénicos/aislamiento & purificación , Antineoplásicos Fitogénicos/farmacología , Aspergillus/metabolismo , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Diterpenos/aislamiento & purificación , Diterpenos/farmacología , HumanosRESUMEN
Studies of the genome-sequenced, flutimide-producing coprophilous fungus Delitschia confertaspora (ATCC 74209), originally obtained from a sample of rock hyrax (Procavia capensis) dung collected in Namibia, led to the discovery of three new highly aromatic natural products named delicoferones A-B (1-2) and fimetarone B (3). The new benzophenone derivatives 1 and 2 have a somewhat unusual skeleton that incorporates three aromatic rings linked via two ketone carbonyl groups, while 3 contains a spiro[chroman-3,7'-isochromene]-4,6'(8'H) skeleton reported only once previously. The structures of these compounds were assigned mainly by analysis of 2D NMR and HRESITOFMS data.
Asunto(s)
Benzofenonas/aislamiento & purificación , Hongos/química , Compuestos de Espiro/aislamiento & purificación , Animales , Benzofenonas/química , Damanes , Estructura Molecular , Namibia , Resonancia Magnética Nuclear Biomolecular , Compuestos de Espiro/químicaRESUMEN
Auxarthrones A-E (1-5), five new phenalenones, and two new naturally occurring cyclic tetrapeptides, auxarthrides A (7) and B (8), were obtained from three different solvent extracts of cultures of the coprophilous fungus Auxarthron pseudauxarthron. Auxarthrones C (3) and E (5) possess an unusual 7a,8-dihydrocyclopenta[a]phenalene-7,9-dione ring system that has not been previously observed in natural products. Formation of 1-5 was found to be dependent on the solvent used for culture extraction. The structures of these new compounds were elucidated primarily by analysis of NMR and MS data. Auxarthrone A (1) was obtained as a mixture of chromatographically inseparable racemic diastereomers (1a and 1b) that cocrystallized, enabling confirmation of their structures by X-ray crystallography. The absolute configurations of 7 and 8 were assigned by analysis of their acid hydrolysates using Marfey's method. Compound 1 displayed moderate antifungal activity against Cryptococcus neoformans and Candida albicans, but did not affect human cancer cell lines.
Asunto(s)
Antifúngicos/aislamiento & purificación , Antifúngicos/farmacología , Ascomicetos/química , Cryptococcus neoformans/efectos de los fármacos , Péptidos Cíclicos/aislamiento & purificación , Péptidos Cíclicos/farmacología , Fenalenos/aislamiento & purificación , Fenalenos/farmacología , Antifúngicos/química , Neoplasias de la Mama/tratamiento farmacológico , Candida albicans/efectos de los fármacos , Cristalografía por Rayos X , Femenino , Humanos , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Péptidos Cíclicos/química , Fenalenos/químicaRESUMEN
Although chronic myelogenous leukemia (CML) is effectively controlled by Bcr-Abl kinase inhibitors, resistance to inhibitors, progressive disease, and incomplete eradication of Bcr-Abl-expressing cells are concerns for the long-term control and suppression of this disease. We describe a novel approach to targeting key proteins in CML cells with a ubiquitin-cycle inhibitor, WP1130. Bcr-Abl is rapidly modified with K63-linked ubiquitin polymers in WP1130-treated CML cells, resulting in its accumulation in aggresomes, where is it unable to conduct signal transduction. Induction of apoptosis because of aggresomal compartmentalization of Bcr-Abl was observed in both imatinib-sensitive and -resistant cells. WP1130, but not Bcr-Abl kinase inhibitors, directly inhibits Usp9x deubiquitinase activity, resulting in the down-regulation of the prosurvival protein Mcl-1 and facilitating apoptosis. These results demonstrate that ubiquitin-cycle inhibition represents a novel and effective approach to blocking Bcr-Abl kinase signaling and reducing Mcl-1 levels to engage CML cell apoptosis. This approach may be a therapeutic option for kinase inhibitor-resistant CML patients.
Asunto(s)
Apoptosis , Proteínas de Fusión bcr-abl/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/enzimología , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Transducción de Señal , Ubiquitina Tiolesterasa/antagonistas & inhibidores , Ubiquitinación , Apoptosis/efectos de los fármacos , Benzamidas , Línea Celular Tumoral , Cianoacrilatos , Resistencia a Antineoplásicos/efectos de los fármacos , Endopeptidasas/metabolismo , Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Humanos , Mesilato de Imatinib , Modelos Biológicos , Nitrilos/farmacología , Fosforilación/efectos de los fármacos , Piperazinas/farmacología , Transporte de Proteínas/efectos de los fármacos , Piridinas/farmacología , Pirimidinas/farmacología , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Especificidad por Sustrato/efectos de los fármacos , Ubiquitina Tiolesterasa/metabolismo , Ubiquitinación/efectos de los fármacosRESUMEN
Epidermal growth factor receptor (EGFR) exists in the nucleus of highly proliferative cells where it functions as a transcription factor. Although EGFR has transactivational activity, it lacks a DNA binding domain and, therefore, may require a DNA binding transcription cofactor for its transcriptional function. Here, we report that EGFR physically interacts with signal transducers and activators of transcription 3 (STAT3) in the nucleus, leading to transcriptional activation of inducible nitric oxide synthase (iNOS). In breast carcinomas, nuclear EGFR positively correlates with iNOS. This study describes a mode of transcriptional control involving cooperated efforts of STAT3 and nuclear EGFR. Our work suggests that the deregulated iNOS/NO pathway may partly contribute to the malignant biology of tumor cells with high levels of nuclear EGFR and STAT3.
Asunto(s)
Núcleo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Receptores ErbB/metabolismo , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico/fisiología , Transactivadores/metabolismo , Animales , Secuencia de Bases , Sitios de Unión/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Células CHO , Línea Celular Tumoral , Núcleo Celular/efectos de los fármacos , Núcleo Celular/ultraestructura , Supervivencia Celular/efectos de los fármacos , Inmunoprecipitación de Cromatina , Cricetinae , Cricetulus , Sinergismo Farmacológico , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/antagonistas & inhibidores , Femenino , Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica , Genes bcl-1/genética , Genes fos/genética , Células HeLa , Humanos , Janus Quinasa 2 , Microscopía Fluorescente , Microscopía Inmunoelectrónica , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo II , Fosforilación/efectos de los fármacos , Pronóstico , Regiones Promotoras Genéticas/genética , Unión Proteica/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , S-Nitroso-N-Acetilpenicilamina/farmacología , Factor de Transcripción STAT3 , Transducción de Señal/efectos de los fármacos , Análisis de SupervivenciaRESUMEN
Triple-negative breast cancer (TNBC) has high relapse and metastasis rates and a high proportion of cancer stem-like cells (CSC), which possess self-renewal and tumor initiation capacity. MELK (maternal embryonic leucine zipper kinase), a protein kinase of the Snf1/AMPK kinase family, is known to promote CSC maintenance and malignant transformation. However, the role of MELK in TNBC metastasis is unknown; we sought to address this in the current study. We found that MELK mRNA levels were higher in TNBC tumors [8.11 (3.79-10.95)] than in HR+HER2- tumors [6.54 (2.90-9.26)]; P < 0.001]. In univariate analysis, patients with breast cancer with high-MELK-expressing tumors had worse overall survival (P < 0.001) and distant metastasis-free survival (P < 0.01) than patients with low-MELK-expressing tumors. In a multicovariate Cox regression model, high MELK expression was associated with shorter overall survival after adjusting for other baseline risk factors. MELK knockdown using siRNA or MELK inhibition using the MELK inhibitor MELK-In-17 significantly reduced invasiveness, reversed epithelial-to-mesenchymal transition, and reduced CSC self-renewal and maintenance in TNBC cells. Nude mice injected with CRISPR MELK-knockout MDA-MB-231 cells exhibited suppression of lung metastasis and improved overall survival compared with mice injected with control cells (P < 0.05). Furthermore, MELK-In-17 suppressed 4T1 tumor growth in syngeneic BALB/c mice (P < 0.001). Our findings indicate that MELK supports metastasis by promoting epithelial-to-mesenchymal transition and the CSC phenotype in TNBC. Significance: These findings indicate that MELK is a driver of aggressiveness and metastasis in TNBC.
Asunto(s)
Neoplasias de la Mama Triple Negativas , Humanos , Animales , Ratones , Neoplasias de la Mama Triple Negativas/genética , Ratones Desnudos , Leucina Zippers , Proliferación Celular/fisiología , Recurrencia Local de Neoplasia , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
Pathological expression of human ErbB-2 protein, also known as HER-2, is common in many types of cancer. ErbB-2 is a member of the EGF receptor tyrosine kinase family and has been rigorously studied as a signaling molecule on the cell membrane. Here, we report that ErbB-2 is also expressed in the nucleus in cultured cells as well as primary tumor tissues. Nuclear ErbB-2 was found to associate with multiple genomic targets in vivo, including the cyclooxygenase enzyme COX-2 gene promoter. ErbB-2 forms a complex at a specific nucleotide sequence of the COX-2 promoter and is able to stimulate its transcription. This study demonstrates the presence of ErbB-2 in the nucleus and identifies the function of ErbB-2 as a transcriptional regulator.
Asunto(s)
Isoenzimas/genética , Prostaglandina-Endoperóxido Sintasas/genética , Receptor ErbB-2/fisiología , Activación Transcripcional , Secuencia de Bases , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/genética , Núcleo Celular/metabolismo , Ciclooxigenasa 2 , Proteínas de Unión al ADN/metabolismo , Humanos , Proteínas de la Membrana , Regiones Promotoras Genéticas , Células Tumorales CultivadasRESUMEN
AIM: Multiple myeloma (MM) is a hematological malignancy of antibody-producing mature B cells or plasma cells. The proteasome inhibitor, bortezomib, was the first-in-class compound to be FDA approved for MM and is frequently utilized in induction therapy. However, bortezomib refractory disease is a major clinical concern, and the efficacy of the pan-histone deacetylase inhibitor (HDACi), panobinostat, in bortezomib refractory disease indicates that HDAC targeting is a viable strategy. Here, we utilized isogenic bortezomib resistant models to profile HDAC expression and define baseline and HDACi-induced expression patterns of individual HDAC family members in sensitive vs. resistant cells to better understanding the potential for targeting these enzymes. METHODS: Gene expression of HDAC family members in two sets of isogenic bortezomib sensitive or resistant myeloma cell lines was examined. These cell lines were subsequently treated with HDAC inhibitors: panobinostat or vorinostat, and HDAC expression was evaluated. CRISPR/Cas9 knockdown and pharmacological inhibition of specific HDAC family members were conducted. RESULTS: Interestingly, HDAC6 and HDAC7 were significantly upregulated and downregulated, respectively, in bortezomib-resistant cells. Panobinostat was effective at inducing cell death in these lines and modulated HDAC expression in cell lines and patient samples. Knockdown of HDAC7 inhibited cell growth while pharmacologically inhibiting HDAC6 augmented cell death by panobinostat. CONCLUSION: Our data revealed heterogeneous expression of individual HDACs in bortezomib sensitive vs. resistant isogenic cell lines and patient samples treated with panobinostat. Cumulatively our findings highlight distinct roles for HDAC6 and HDAC7 in regulating cell death in the context of bortezomib resistance.
RESUMEN
Myelofibrosis (MF) is a myeloproliferative neoplasm characterized by cytopenia and extramedullary hematopoiesis, resulting in splenomegaly. Multiple pathological mechanisms (e.g., circulating cytokines and genetic alterations, such as JAKV617F mutation) have been implicated in the etiology of MF, but the molecular mechanism causing resistance to JAK2V617F inhibitor therapy remains unknown. Among MF patients who were treated with the JAK inhibitor ruxolitinib, we compared noncoding RNA profiles of ruxolitinib therapy responders versus nonresponders and found miR-543 was significantly upregulated in nonresponders. We validated these findings by reverse transcription-quantitative PCR. in this same cohort, in 2 additional independent MF patient cohorts from the United States and Romania, and in a JAK2V617F mouse model of MF. Both in vitro and in vivo models were used to determine the underlying molecular mechanism of miR-543 in MF. Here, we demonstrate that miR-543 targets the dioxygenases ten-eleven translocation 1 (TET1) and 2 (TET2) in patients and in vitro, causing increased levels of global 5-methylcytosine, while decreasing the acetylation of histone 3, STAT3, and tumor protein p53. Mechanistically, we found that activation of STAT3 by JAKs epigenetically controls miR-543 expression via binding the promoter region of miR-543. Furthermore, miR-543 upregulation promotes the expression of genes related to drug metabolism, including CYP3A4, which is involved in ruxolitinib metabolism. Our findings suggest miR-543 as a potentially novel biomarker for the prognosis of MF patients with a high risk of treatment resistance and as a potentially new target for the development of new treatment options.
Asunto(s)
Proteínas de Unión al ADN/efectos de los fármacos , Epigénesis Genética/efectos de los fármacos , MicroARNs/metabolismo , MicroARNs/farmacología , Mielofibrosis Primaria/tratamiento farmacológico , Proteínas Proto-Oncogénicas/efectos de los fármacos , Animales , Citocinas/metabolismo , Proteínas de Unión al ADN/genética , Dioxigenasas , Modelos Animales de Enfermedad , Histonas , Humanos , Inhibidores de las Cinasas Janus/uso terapéutico , Quinasas Janus/metabolismo , Ratones , MicroARNs/genética , Oxigenasas de Función Mixta , Mutación , Trastornos Mieloproliferativos , Nitrilos , Mielofibrosis Primaria/genética , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas/genética , Pirazoles/uso terapéutico , Pirimidinas , Factor de Transcripción STAT3 , Transcriptoma , Estados UnidosRESUMEN
c-Myc is a highly unstable transcription factor whose deregulation and increased expression are associated with cancer. Degrasyn, a small synthetic molecule, induces rapid degradation of c-Myc protein in MM-1 multiple myeloma and other tumor cell lines. Destruction of c-Myc by degrasyn requires the presence of a region of c-Myc between amino acid residues 316 and 378 that has not previously been associated with c-Myc stability. Degrasyn-induced degradation of c-Myc depends on proteasomes but is independent of the degron regions previously shown to be important for ubiquitin-mediated targeting and proteasomal destruction of the protein. Degrasyn-dependent c-Myc proteolysis is not mediated by any previously identified c-Myc regulatory mechanism, does not require new protein synthesis, and does not depend on the nuclear localization of c-Myc. Degrasyn reduced c-Myc levels in A375 melanoma cells and in A375 tumors in nude mice, and this activity correlated with tumor growth inhibition. Together, these results suggest that degrasyn reduces the stability of c-Myc in vitro and in vivo through a unique signaling process that uses c-Myc domains not previously associated with c-Myc regulation.
Asunto(s)
Melanoma/tratamiento farmacológico , Nitrilos/farmacología , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Piridinas/farmacología , Animales , Línea Celular Tumoral , Cianoacrilatos , Regulación hacia Abajo/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Femenino , Células HeLa , Humanos , Melanoma/enzimología , Melanoma/metabolismo , Ratones , Ratones Desnudos , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/enzimología , Mieloma Múltiple/metabolismo , Mapeo Peptídico , Estructura Terciaria de Proteína , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
For mucinous ovarian cancer (MOC), standard platinum-based therapy is largely ineffective. We sought to identify possible mechanisms of oxaliplatin resistance of MOC and develop strategies to overcome this resistance. A kinome-based siRNA library screen was carried out using human MOC cells to identify novel targets to enhance the efficacy of chemotherapy. In vitro and in vivo validations of antitumor effects were performed using mouse MOC models. Specifically, the role of PRKRA/PACT in oxaliplatin resistance was interrogated. We focused on PRKRA, a known activator of PKR kinase, and its encoded protein PACT because it was one of the five most significantly downregulated genes in the siRNA screen. In orthotopic mouse models of MOC, we observed a significant antitumor effect of PRKRA siRNA plus oxaliplatin. In addition, expression of miR-515-3p was regulated by PACT-Dicer interaction, and miR-515-3p increased the sensitivity of MOC to oxaliplatin. Mechanistically, miR-515-3p regulated chemosensitivity, in part, by targeting AXL. The PRKRA/PACT axis represents an important therapeutic target in MOC to enhance sensitivity to oxaliplatin.
Asunto(s)
Adenocarcinoma Mucinoso/patología , Resistencia a Antineoplásicos , Neoplasias Ováricas/patología , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Regulación hacia Arriba , Adenocarcinoma Mucinoso/genética , Adenocarcinoma Mucinoso/metabolismo , Animales , Línea Celular Tumoral , Supervivencia Celular , ARN Helicasas DEAD-box/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Ratones , MicroARNs/genética , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Oxaliplatino , Proteínas Proto-Oncogénicas/genética , ARN Interferente Pequeño/farmacología , Proteínas Tirosina Quinasas Receptoras/genética , Ribonucleasa III/metabolismo , Tirosina Quinasa del Receptor AxlRESUMEN
PURPOSE: Paclitaxel is an integral component of primary therapy for breast and epithelial ovarian cancers, but less than half of these cancers respond to the drug. Enhancing the response to primary therapy with paclitaxel could improve outcomes for women with both diseases.Experimental Design: Twelve kinases that regulate metabolism were depleted in multiple ovarian and breast cancer cell lines to determine whether they regulate sensitivity to paclitaxel in Sulforhodamine B assays. The effects of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 2 (PFKFB2) depletion on cell metabolomics, extracellular acidification rate, nicotinamide adenine dinucleotide phosphate, reactive oxygen species (ROS), and apoptosis were studied in multiple ovarian and breast cancer cell lines. Four breast and ovarian human xenografts and a breast cancer patient-derived xenograft (PDX) were used to examine the knockdown effect of PFKFB2 on tumor cell growth in vivo. RESULTS: Knockdown of PFKFB2 inhibited clonogenic growth and enhanced paclitaxel sensitivity in ovarian and breast cancer cell lines with wild-type TP53 (wtTP53). Silencing PFKFB2 significantly inhibited tumor growth and enhanced paclitaxel sensitivity in four xenografts derived from two ovarian and two breast cancer cell lines, and prolonged survival in a triple-negative breast cancer PDX. Transfection of siPFKFB2 increased the glycolysis rate, but decreased the flow of intermediates through the pentose-phosphate pathway in cancer cells with wtTP53, decreasing NADPH. ROS accumulated after PFKFB2 knockdown, which stimulated Jun N-terminal kinase and p53 phosphorylation, and induced apoptosis that depended upon upregulation of p21 and Puma. CONCLUSIONS: PFKFB2 is a novel target whose inhibition can enhance the effect of paclitaxel-based primary chemotherapy upon ovarian and breast cancers retaining wtTP53.
Asunto(s)
Neoplasias de la Mama/metabolismo , Resistencia a Antineoplásicos , Neoplasias Ováricas/metabolismo , Paclitaxel/farmacología , Fosfofructoquinasa-2/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos/genética , Femenino , Expresión Génica , Silenciador del Gen , Humanos , Inmunohistoquímica , Redes y Vías Metabólicas , Ratones , Mutación , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Estrés Oxidativo , Fosfofructoquinasa-2/genética , Interferencia de ARN , Especies Reactivas de Oxígeno/metabolismo , Proteína p53 Supresora de Tumor/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The cell membrane receptor ErbB-2 migrates to the nucleus. However, the mechanism of its nuclear translocation is unclear. Here, we report a novel mechanism of its nuclear localization that involves interaction with the transport receptor importin beta1, nuclear pore protein Nup358, and a host of players in endocytic internalization. Knocking down importin beta1 using small interfering RNA oligonucleotides or inactivation of small GTPase Ran by RanQ69L, a dominant-negative mutant of Ran, causes a nuclear transport defect of ErbB-2. Mutation of a putative nuclear localization signal in ErbB-2 destroys its interaction with importin beta1 and arrests nuclear translocation, while inactivation of nuclear export receptor piles up ErbB-2 within the nucleus. Additionally, blocking of internalization by a dominant-negative mutant of dynamin halts its nuclear localization. Thus, the cell membrane-embedded ErbB-2, through endocytosis using the endocytic vesicle as a vehicle, importin beta1 as a driver and Nup358 as a traffic light, migrates from the cell surface to the nucleus. This novel mechanism explains how a receptor tyrosine kinase on the cell surface can be translocated into the nucleus. This pathway may serve as a general mechanism to allow direct communication between cell surface receptors and the nucleus, and our findings thus open a new era in understanding direct trafficking between the cell membrane and nucleus.
Asunto(s)
Núcleo Celular/metabolismo , Endocitosis , Chaperonas Moleculares/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Receptor ErbB-2/metabolismo , Receptores de Superficie Celular/metabolismo , beta Carioferinas/metabolismo , Transporte Activo de Núcleo Celular , Núcleo Celular/química , Células Cultivadas , Clatrina/metabolismo , Endosomas/metabolismo , Humanos , Carioferinas/genética , Carioferinas/metabolismo , Mutación , Señales de Localización Nuclear/genética , Señales de Localización Nuclear/metabolismo , Receptor ErbB-2/análisis , Receptores de Superficie Celular/análisis , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , beta Carioferinas/análisis , Proteína de Unión al GTP ran/genética , Proteína de Unión al GTP ran/metabolismo , Proteína Exportina 1RESUMEN
Inflammatory breast cancer (IBC) is a rare and aggressive presentation of invasive breast cancer with a 62% to 68% 5-year survival rate. It is the most lethal form of breast cancer, and early recognition and treatment is important for patient survival. Like non-inflammatory breast cancer, IBC comprises multiple subtypes, with the triple-negative subtype being overrepresented. Although the current multimodality treatment regime of anthracycline- and taxane-based neoadjuvant therapy, surgery, and radiotherapy has improved the outcome of patients with triple-negative IBC, overall survival continues to be worse than in patients with non-inflammatory locally advanced breast cancer. Translation of new therapies into the clinics to successfully treat IBC has been poor, in part because of the lack of in vitro preclinical models that can accurately predict the response of the original tumor to therapy. We report the generation of a preclinical IBC patient-derived xenograft (PDX)-derived ex vivo (PDXEx) model and show that it closely replicates the tissue architecture of the original PDX tumor harvested from mice. The gene expression profile of our IBC PDXEx model had a high degree of correlation to that of the original tumor. This suggests that the process of generating the PDXEx model did not significantly alter the molecular signature of the original tumor. We demonstrate a high degree of similarity in drug response profile between a PDX mouse model and our PDXEx model generated from the same original PDX tumor tissue and treated with the same panel of drugs, indicating that our PDXEx model had high predictive value in identifying effective tumor-specific therapies. Finally, we used our PDXEx model as a platform for a robotic-based high-throughput drug screen of a 386-drug anti-cancer compound library. The top candidates identified from this drug screen all demonstrated greater therapeutic efficacy than the standard-of-care drugs used in the clinic to treat triple-negative IBC, doxorubicin and paclitaxel. Our PDXEx model is simple, and we are confident that it can be incorporated into a PDX mouse system for use as a first-pass screening platform. This will permit the identification of effective tumor-specific therapies with high predictive value in a resource-, time-, and cost-efficient manner.