RESUMEN
Platinum-based combination chemotherapy is the standard treatment for advanced non-small cell lung cancer (NSCLC). While cisplatin is effective, its use is not curative and resistance often emerges. As a consequence of microenvironmental heterogeneity, many tumour cells are exposed to sub-lethal doses of cisplatin. Further, genomic heterogeneity and unique tumor cell sub-populations with reduced sensitivities to cisplatin play a role in its effectiveness within a site of tumor growth. Being exposed to sub-lethal doses will induce changes in gene expression that contribute to the tumour cell's ability to survive and eventually contribute to the selective pressures leading to cisplatin resistance. Such changes in gene expression, therefore, may contribute to cytoprotective mechanisms. Here, we report on studies designed to uncover how tumour cells respond to sub-lethal doses of cisplatin. A microarray study revealed changes in gene expressions that occurred when A549 cells were exposed to a no-observed-effect level (NOEL) of cisplatin (e.g. the IC10). These data were integrated with results from a genome-wide siRNA screen looking for novel therapeutic targets that when inhibited transformed a NOEL of cisplatin into one that induced significant increases in lethality. Pathway analyses were performed to identify pathways that could be targeted to enhance cisplatin activity. We found that over 100 genes were differentially expressed when A549 cells were exposed to a NOEL of cisplatin. Pathways associated with apoptosis and DNA repair were activated. The siRNA screen revealed the importance of the hedgehog, cell cycle regulation, and insulin action pathways in A549 cell survival and response to cisplatin treatment. Results from both datasets suggest that RRM2B, CABYR, ALDH3A1, and FHL2 could be further explored as cisplatin-enhancing gene targets. Finally, pathways involved in repairing double-strand DNA breaks and INO80 chromatin remodeling were enriched in both datasets, warranting further research into combinations of cisplatin and therapeutics targeting these pathways.
Asunto(s)
Antineoplásicos/química , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Cisplatino/química , Resistencia a Antineoplásicos , Neoplasias Pulmonares/metabolismo , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al Calcio/genética , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral/efectos de los fármacos , Cromatina/metabolismo , Roturas del ADN de Doble Cadena , Reparación del ADN , Ensayos de Selección de Medicamentos Antitumorales , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Humanos , Fosfoproteínas/genética , Ribonucleótido Reductasas/genéticaRESUMEN
Standard treatment for advanced non-small cell lung cancer (NSCLC) with no known driver mutation is platinum-based chemotherapy, which has a response rate of only 30-33%. Through an siRNA screen, 3'-phosphoadenosine 5'-phosphosulfate (PAPS) synthase 1 (PAPSS1), an enzyme that synthesizes the biologically active form of sulfate PAPS, was identified as a novel platinum-sensitizing target in NSCLC cells. PAPSS1 knockdown in combination with low-dose (IC10) cisplatin reduces clonogenicity of NSCLC cells by 98.7% (p < 0.001), increases DNA damage, and induces G1/S phase cell cycle arrest and apoptosis. PAPSS1 silencing also sensitized NSCLC cells to other DNA crosslinking agents, radiation, and topoisomerase I inhibitors, but not topoisomerase II inhibitors. Chemo-sensitization was not observed in normal epithelial cells. Knocking out the PAPSS1 homolog did not sensitize yeast to cisplatin, suggesting that sulfate bioavailability for amino acid synthesis is not the cause of sensitization to DNA damaging agents. Rather, sensitization may be due to sulfation reactions involved in blocking the action of DNA damaging agents, facilitating DNA repair, promoting cancer cell survival under therapeutic stress or reducing the bioavailability of DNA damaging agents. Our study demonstrates for the first time that PAPSS1 could be targeted to improve the activity of multiple anticancer agents used to treat NSCLC.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/genética , Resistencia a Antineoplásicos/genética , Neoplasias Pulmonares/genética , Complejos Multienzimáticos/genética , Sulfato Adenililtransferasa/genética , Antineoplásicos/farmacología , Western Blotting , Carcinoma de Pulmón de Células no Pequeñas/enzimología , Línea Celular Tumoral , Cisplatino/farmacología , Daño del ADN/efectos de los fármacos , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Técnicas de Silenciamiento del Gen , Humanos , Neoplasias Pulmonares/enzimología , Complejos Multienzimáticos/metabolismo , ARN Interferente Pequeño , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sulfato Adenililtransferasa/metabolismo , TransfecciónRESUMEN
Childhood sarcomas can be extremely difficult to accurately diagnose on the basis of morphological characteristics alone. Ancillary methods, such as RT-PCR or fluorescence in situ hybridization, to detect pathognomonic gene fusions can help to distinguish these tumors. Two major deficiencies of these assays are their inability to identify gene fusions at nucleotide resolution or to detect multiple gene fusions simultaneously. We developed a next-generation sequencing-based assay designated ChildSeq-RNA that uses the Ion Torrent platform to screen for EWSR1-FLI1 and EWSR1-ERG, PAX3-FOXO1 and PAX7-FOXO1, EWSR1-WT1, and ETV6-NTRK3 fusions of Ewing sarcoma (ES), alveolar rhabdomyosarcoma, desmoplastic small round cell tumor, and congenital fibrosarcoma, respectively. To rapidly analyze resulting data, we codeveloped a bioinformatics tool, termed ChildDecode, that operates on a scalable, cloud-computing platform. Total RNA from four ES cell lines plus 33 clinical samples representing ES, alveolar rhabdomyosarcoma, desmoplastic small round cell tumor, and congenital fibrosarcoma tumors was subjected to ChildSeq-RNA. This accurately identified corresponding gene fusions in each tumor type, with no examples of false positive fusion detection in this proof-of-concept study. Comparison with previous RT-PCR findings demonstrated high sensitivity (96.4%; 95% CI, 82.3%-99.4%) and specificity (100%; 95% CI, 56.6%-100%) of ChildSeq-RNA to detect gene fusions. Herein, we propose ChildSeq-RNA as a novel tool to detect gene fusions in childhood sarcomas at single-nucleotide resolution.
Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Proteínas de Fusión Oncogénica/genética , Sarcoma/genética , Análisis de Secuencia de ARN/métodos , Línea Celular Tumoral , Niño , Humanos , Fusión de Oncogenes/genética , Factores de Transcripción Paired Box/genética , ARN/genética , Sensibilidad y EspecificidadRESUMEN
Gefitinib (Iressa(®), ZD1839) is a small molecule inhibitor of the epidermal growth factor receptor (EGFR) tyrosine kinase. We report on an early cellular response to gefitinib that involves induction of functional autophagic flux in phenotypically diverse breast cancer cells that were sensitive (BT474 and SKBR3) or insensitive (MCF7-GFPLC3 and JIMT-1) to gefitinib. Our data show that elevation of autophagy in gefitinib-treated breast cancer cells correlated with downregulation of AKT and ERK1/2 signaling early in the course of treatment. Inhibition of autophagosome formation by BECLIN-1 or ATG7 siRNA in combination with gefitinib reduced the abundance of autophagic organelles and sensitized SKBR3 but not MCF7-GFPLC3 cells to cell death. However, inhibition of the late stage of gefitinib-induced autophagy with hydroxychloroquine (HCQ) or bafilomycin A1 significantly increased (p<0.05) cell death in gefitinib-sensitive SKBR3 and BT474 cells, as well as in gefitinib-insensitive JIMT-1 and MCF7-GFPLC3 cells, relative to the effects observed with the respective single agents. Treatment with the combination of gefitinib and HCQ was more effective (p<0.05) in delaying tumor growth than either monotherapy (p>0.05), when compared to vehicle-treated controls. Our results also show that elevated autophagosome content following short-term treatment with gefitinib is a reversible response that ceases upon removal of the drug. In aggregate, these data demonstrate that elevated autophagic flux is an early response to gefitinib and that targeting EGFR and autophagy should be considered when developing new therapeutic strategies for EGFR expressing breast cancers.
Asunto(s)
Autofagia/efectos de los fármacos , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Quinazolinas/farmacología , Quinazolinas/uso terapéutico , Adenina/análogos & derivados , Adenina/farmacología , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 7 Relacionada con la Autofagia , Beclina-1 , Neoplasias de la Mama/ultraestructura , Cadaverina/análogos & derivados , Cadaverina/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Vesículas Citoplasmáticas/efectos de los fármacos , Vesículas Citoplasmáticas/metabolismo , Vesículas Citoplasmáticas/ultraestructura , Receptores ErbB/metabolismo , Femenino , Gefitinib , Técnicas de Silenciamiento del Gen , Silenciador del Gen/efectos de los fármacos , Humanos , Hidroxicloroquina/farmacología , Hidroxicloroquina/uso terapéutico , Proteínas de la Membrana/metabolismo , Ratones , Fagosomas/efectos de los fármacos , Fagosomas/metabolismo , Fagosomas/ultraestructura , ARN Interferente Pequeño/metabolismo , Transducción de Señal/efectos de los fármacos , Coloración y Etiquetado , Tamoxifeno/farmacología , Tamoxifeno/uso terapéutico , Resultado del Tratamiento , Enzimas Activadoras de Ubiquitina/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: HER2-positive breast cancers exhibit high rates of innate and acquired resistance to trastuzumab (TZ), a HER2-directed antibody used as a first line treatment for this disease. TZ resistance may in part be mediated by frequent co-expression of EGFR and by sustained activation of the mammalian target of rapamycin (mTOR) pathway. Here, we assessed feasibility of combining the EGFR inhibitor gefitinib and the mTOR inhibitor everolimus (RAD001) for treating HER2 overexpressing breast cancers with different sensitivity to TZ. METHODS: The gefitinib and RAD001 combination was broadly evaluated in TZ sensitive (SKBR3 and MCF7-HER2) and TZ resistant (JIMT-1) breast cancer models. The effects on cell growth were measured in cell based assays using the fixed molar ratio design and the median effect principle. In vivo studies were performed in Rag2M mice bearing established tumors. Analysis of cell cycle, changes in targeted signaling pathways and tumor characteristics were conducted to assess gefitinib and RAD001 interactions. RESULTS: The gefitinib and RAD001 combination inhibited cell growth in vitro in a synergistic fashion as defined by the Chou and Talalay median effect principle and increased tumor xenograft growth delay. The improvement in therapeutic efficacy by the combination was associated in vitro with cell line dependent increases in cytotoxicity and cytostasis while treatment in vivo promoted cytostasis. The most striking and consistent therapeutic effect of the combination was increased inhibition of the mTOR pathway (in vitro and in vivo) and EGFR signaling in vivo relative to the single drugs. CONCLUSIONS: The gefitinib and RAD001 combination provides effective control over growth of HER2 overexpressing cells and tumors irrespective of the TZ sensitivity status.
Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Quinazolinas/uso terapéutico , Receptor ErbB-2/genética , Sirolimus/análogos & derivados , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias de la Mama/genética , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Sinergismo Farmacológico , Receptores ErbB/antagonistas & inhibidores , Everolimus , Femenino , Gefitinib , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Fosforilación/efectos de los fármacos , Quinazolinas/administración & dosificación , Receptor ErbB-2/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Sirolimus/administración & dosificación , Sirolimus/uso terapéutico , Serina-Treonina Quinasas TOR/metabolismo , Trastuzumab , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
A gene encoding a putative homologue of the avian and mammalian vascular smooth muscle alpha-actin was isolated from an amphibian, Rana catesbeiana, and characterized in terms of its sequence, organization, and expression pattern. To assess the expression of this gene during amphibian embryonic development, a cDNA encoding the Xenopus homologue of this mRNA was isolated and characterized by in situ hybridization. The expression of this gene was not detected in the enteric smooth muscle cells or, unlike its avian and mammalian homologues, in the somites/skeletal muscle of the Xenopus embryos/tadpoles. Its initial expression coincides with the onset of cardiac muscle differentiation and is coincidental with the expression of the cardiac alpha-actin mRNAs in the heart-forming region of the stage 26/27 embryo. As development proceeds, transcripts from this gene are expressed throughout the developing heart until the formation of the heart chambers is completed and, thereafter, its expression becomes restricted to the outflow tract of the tadpole heart. The subsequent restricted expression of this gene to the vascular system in both of these amphibians identifies it as the amphibian homologue of the avian and mammalian vascular smooth muscle alpha-actin.