RESUMEN
Gene expression by RNA polymerase II (RNAPII) is tightly controlled by cyclin-dependent kinases (CDKs) at discrete checkpoints during the transcription cycle. The pausing checkpoint following transcription initiation is primarily controlled by CDK9. We discovered that CDK9-mediated, RNAPII-driven transcription is functionally opposed by a protein phosphatase 2A (PP2A) complex that is recruited to transcription sites by the Integrator complex subunit INTS6. PP2A dynamically antagonizes phosphorylation of key CDK9 substrates including DSIF and RNAPII-CTD. Loss of INTS6 results in resistance to tumor cell death mediated by CDK9 inhibition, decreased turnover of CDK9 phospho-substrates, and amplification of acute oncogenic transcriptional responses. Pharmacological PP2A activation synergizes with CDK9 inhibition to kill both leukemic and solid tumor cells, providing therapeutic benefit in vivo. These data demonstrate that fine control of gene expression relies on the balance between kinase and phosphatase activity throughout the transcription cycle, a process dysregulated in cancer that can be exploited therapeutically.
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Quinasa 9 Dependiente de la Ciclina/metabolismo , Terapia Molecular Dirigida , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Proteína Fosfatasa 2/metabolismo , Proteínas de Unión al ARN/metabolismo , Transcripción Genética , Proteínas Supresoras de Tumor/metabolismo , Animales , Línea Celular Tumoral , Quinasa 9 Dependiente de la Ciclina/antagonistas & inhibidores , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones Endogámicos NOD , Fosforilación , Unión Proteica , ARN Polimerasa II/química , ARN Polimerasa II/metabolismo , Especificidad por SustratoRESUMEN
There are diverse phenotypes of castration-resistant prostate cancer, including neuroendocrine disease, that vary in their sensitivity to drug treatment. The efficacy of BET and CBP/p300 inhibitors in prostate cancer is attributed, at least in part, to their ability to decrease androgen receptor (AR) signalling. However, the activity of BET and CBP/p300 inhibitors in prostate cancers that lack the AR is unclear. In this study, we showed that BRD4, CBP, and p300 were co-expressed in AR-positive and AR-null prostate cancer. A combined inhibitor of these three proteins, NEO2734, reduced the growth of both AR-positive and AR-null organoids, as measured by changes in viability, size, and composition. NEO2734 treatment caused consistent transcriptional downregulation of cell cycle pathways. In neuroendocrine models, NEO2734 treatment reduced ASCL1 levels and other neuroendocrine markers, and reduced tumour growth in vivo. Collectively, these results show that epigenome-targeted inhibitors cause decreased growth and phenotype-dependent disruption of lineage regulators in neuroendocrine prostate cancer, warranting further development of compounds with this activity in the clinic. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
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Proteína p300 Asociada a E1A , Receptores Androgénicos , Transducción de Señal , Masculino , Humanos , Receptores Androgénicos/metabolismo , Receptores Androgénicos/genética , Animales , Proteína p300 Asociada a E1A/metabolismo , Proteína p300 Asociada a E1A/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Neoplasias de la Próstata Resistentes a la Castración/patología , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Neoplasias de la Próstata Resistentes a la Castración/genética , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/genética , Regulación Neoplásica de la Expresión Génica , Ratones , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas que Contienen Bromodominio , Proteína de Unión a CREBRESUMEN
BACKGROUND: Cyclin-dependent kinase 9 (CDK9) stimulates oncogenic transcriptional pathways in cancer and CDK9 inhibitors have emerged as promising therapeutic candidates. METHODS: The activity of an orally bioavailable CDK9 inhibitor, CDKI-73, was evaluated in prostate cancer cell lines, a xenograft mouse model, and patient-derived tumor explants and organoids. Expression of CDK9 was evaluated in clinical specimens by mining public datasets and immunohistochemistry. Effects of CDKI-73 on prostate cancer cells were determined by cell-based assays, molecular profiling and transcriptomic/epigenomic approaches. RESULTS: CDKI-73 inhibited proliferation and enhanced cell death in diverse in vitro and in vivo models of androgen receptor (AR)-driven and AR-independent models. Mechanistically, CDKI-73-mediated inhibition of RNA polymerase II serine 2 phosphorylation resulted in reduced expression of BCL-2 anti-apoptotic factors and transcriptional defects. Transcriptomic and epigenomic approaches revealed that CDKI-73 suppressed signaling pathways regulated by AR, MYC, and BRD4, key drivers of dysregulated transcription in prostate cancer, and reprogrammed cancer-associated super-enhancers. These latter findings prompted the evaluation of CDKI-73 with the BRD4 inhibitor AZD5153, a combination that was synergistic in patient-derived organoids and in vivo. CONCLUSION: Our work demonstrates that CDK9 inhibition disrupts multiple oncogenic pathways and positions CDKI-73 as a promising therapeutic agent for prostate cancer, particularly aggressive, therapy-resistant subtypes.
RESUMEN
Neuroblastoma is a deadly childhood cancer arising in the developing sympathetic nervous system. High-risk patients are currently treated with intensive chemotherapy, which is curative in only 50% of children and leaves some surviving patients with life-long side effects. microRNAs (miRNAs) are critical regulators of neural crest development and are deregulated during neuroblastoma tumorigenesis, making miRNA-based drugs an attractive therapeutic avenue. A functional screen of >1,200 miRNA mimics was conducted in neuroblastoma cell lines to discover miRNAs that sensitized cells to low doses (30% inhibitory concentration [IC30]) of doxorubicin and vincristine chemotherapy used in the treatment of the disease. Three miRNAs, miR-99b-5p, miR-380-3p, and miR-485-3p, had potent chemosensitizing activity with doxorubicin in multiple models of high-risk neuroblastoma. These miRNAs underwent genomic loss in a subset of neuroblastoma patients, and low expression predicted poor survival outcome. In vitro functional assays revealed each of these miRNAs enhanced the anti-proliferative and pro-apoptotic effects of doxorubicin. We used RNA sequencing (RNA-seq) to show that miR-99b-5p represses neuroblastoma dependency genes LIN28B and PHOX2B both in vitro and in patient-derived xenograft (PDX) tumors. Luciferase reporter assays demonstrate that PHOX2B is a direct target of miR-99b-5p. We anticipate that restoring the function of the tumor-suppressive miRNAs discovered here may be a valuable therapeutic strategy for the treatment of neuroblastoma patients.
Asunto(s)
MicroARNs , Neuroblastoma , Niño , Doxorrubicina/farmacología , Doxorrubicina/uso terapéutico , Regulación Neoplásica de la Expresión Génica , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/genéticaRESUMEN
Coxiella burnetii is an intracellular pathogen that replicates in a lysosome-like vacuole through activation of a Dot/Icm-type IVB secretion system and subsequent translocation of effectors that remodel the host cell. Here a genome-wide small interfering RNA screen and reporter assay were used to identify host proteins required for Dot/Icm effector translocation. Significant, and independently validated, hits demonstrated the importance of multiple protein families required for endocytic trafficking of the C. burnetii-containing vacuole to the lysosome. Further analysis demonstrated that the degradative activity of the lysosome created by proteases, such as TPP1, which are transported to the lysosome by receptors, such as M6PR and LRP1, are critical for C. burnetii virulence. Indeed, the C. burnetii PmrA/B regulon, responsible for transcriptional up-regulation of genes encoding the Dot/Icm apparatus and a subset of effectors, induced expression of a virulence-associated transcriptome in response to degradative products of the lysosome. Luciferase reporter strains, and subsequent RNA-sequencing analysis, demonstrated that particular amino acids activate the C. burnetii PmrA/B two-component system. This study has further enhanced our understanding of C. burnetii pathogenesis, the host-pathogen interactions that contribute to bacterial virulence, and the different environmental triggers pathogens can sense to facilitate virulence.
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Proteínas Bacterianas/metabolismo , Sistemas de Secreción Bacterianos/fisiología , Coxiella burnetii/fisiología , Interacciones Huésped-Patógeno , Lisosomas/metabolismo , Fiebre Q/microbiología , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Células HeLa , Humanos , Lisosomas/microbiología , Transporte de Proteínas , Tripeptidil Peptidasa 1 , VirulenciaRESUMEN
Alternative polyadenylation (APA) determines stability, localization and translation potential of the majority of mRNA in eukaryotic cells. The heterodimeric mammalian cleavage factor II (CF IIm) is required for pre-mRNA 3' end cleavage and is composed of the RNA kinase hClp1 and the termination factor hPcf11; the latter protein binds to RNA and the RNA polymerase II carboxy-terminal domain. Here, we used siRNA mediated knockdown and poly(A) targeted RNA sequencing to analyze the role of CF IIm in gene expression and APA in estrogen receptor positive MCF7 breast cancer cells. Identified gene ontology terms link CF IIm function to regulation of growth factor activity, protein heterodimerization and the cell cycle. An overlapping requirement for hClp1 and hPcf11 suggested that CF IIm protein complex was involved in the selection of proximal poly(A) sites. In addition to APA shifts within 3' untranslated regions (3'-UTRs), we observed shifts from promoter proximal regions to the 3'-UTR facilitating synthesis of full-length mRNAs. Moreover, we show that several truncated mRNAs that resulted from APA within introns in MCF7 cells cosedimented with ribosomal components in an EDTA sensitive manner suggesting that those are translated into protein. We propose that CF IIm contributes to the regulation of mRNA function in breast cancer.
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Neoplasias de la Mama/genética , Poliadenilación/genética , Factores de Escisión y Poliadenilación de ARNm/genética , Regiones no Traducidas 3'/genética , Línea Celular Tumoral , Humanos , Células MCF-7 , Poli A/genética , Unión Proteica/genética , ARN Polimerasa II/genética , Precursores del ARN/genética , ARN Mensajero/genéticaRESUMEN
The Dot/Icm system of Legionella pneumophila is essential for virulence and delivers a large repertoire of effectors into infected host cells to create the Legionella containing vacuole. Since the secretion of effectors via the Dot/Icm system does not occur in the absence of host cells, we hypothesised that host factors actively participate in Dot/Icm effector translocation. Here we employed a high-throughput, genome-wide siRNA screen to systematically test the effect of silencing 18,120 human genes on translocation of the Dot/Icm effector, RalF, into HeLa cells. For the primary screen, we found that silencing of 119 genes led to increased translocation of RalF, while silencing of 321 genes resulted in decreased translocation. Following secondary screening, 70 genes were successfully validated as 'high confidence' targets. Gene set enrichment analysis of siRNAs leading to decreased RalF translocation, showed that ubiquitination was the most highly overrepresented category in the pathway analysis. We further showed that two host factors, the E2 ubiquitin-conjugating enzyme, UBE2E1, and the E3 ubiquitin ligase, CUL7, were important for supporting Dot/Icm translocation and L. pneumophila intracellular replication. In summary, we identified host ubiquitin pathways as important for the efficiency of Dot/Icm effector translocation by L. pneumophila, suggesting that host-derived ubiquitin-conjugating enzymes and ubiquitin ligases participate in the translocation of Legionella effector proteins and influence intracellular persistence and survival.
Asunto(s)
Legionella pneumophila , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Células HeLa , Humanos , Legionella pneumophila/genética , Legionella pneumophila/metabolismo , Ubiquitinación , Vacuolas/metabolismoRESUMEN
BACKGROUND: Intrinsic and acquired drug resistance represent fundamental barriers to the cure of high-grade serous ovarian carcinoma (HGSC), the most common histological subtype accounting for the majority of ovarian cancer deaths. Defects in homologous recombination (HR) DNA repair are key determinants of sensitivity to chemotherapy and poly-ADP ribose polymerase inhibitors. Restoration of HR is a common mechanism of acquired resistance that results in patient mortality, highlighting the need to identify new therapies targeting HR-proficient disease. We have shown promise for CX-5461, a cancer therapeutic in early phase clinical trials, in treating HR-deficient HGSC. METHODS: Herein, we screen the whole protein-coding genome to identify potential targets whose depletion cooperates with CX-5461 in HR-proficient HGSC. RESULTS: We demonstrate robust proliferation inhibition in cells depleted of DNA topoisomerase 1 (TOP1). Combining the clinically used TOP1 inhibitor topotecan with CX-5461 potentiates a G2/M cell cycle checkpoint arrest in multiple HR-proficient HGSC cell lines. The combination enhances a nucleolar DNA damage response and global replication stress without increasing DNA strand breakage, significantly reducing clonogenic survival and tumour growth in vivo. CONCLUSIONS: Our findings highlight the possibility of exploiting TOP1 inhibition to be combined with CX-5461 as a non-genotoxic approach in targeting HR-proficient HGSC.
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Benzotiazoles/farmacología , Cistadenocarcinoma Seroso/tratamiento farmacológico , Daño del ADN/efectos de los fármacos , Recombinación Homóloga , Naftiridinas/farmacología , Neoplasias Ováricas/tratamiento farmacológico , ARN Polimerasa I/antagonistas & inhibidores , Inhibidores de Topoisomerasa I/farmacología , Topotecan/farmacología , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/patología , Replicación del ADN/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Sinergismo Farmacológico , Quimioterapia Combinada , Femenino , Puntos de Control de la Fase G1 del Ciclo Celular , Genes BRCA2 , Humanos , Puntos de Control de la Fase M del Ciclo Celular , Ratones , Ratones Endogámicos NOD , Ratones SCID , Clasificación del Tumor , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Interferencia de ARN , ARN Polimerasa I/genéticaRESUMEN
Efficacious therapeutic approaches are urgently needed to improve outcomes in patients with oesophageal adenocarcinoma (OAC). However, oncogenic drivers amenable to targeted therapy are limited and their functional characterisation is essential. Among few targeted therapies available, anti-human epidermal growth factor receptor 2 (HER2) therapy showed only modest benefit for patients with OAC. Herein, we investigated the potential oncogenic role of growth factor receptor bound protein 7 (GRB7), which is reported to be co-amplified with HER2 (ERBB2) in OAC. GRB7 was highly expressed in 15% of OAC tumours, not all of which could be explained by co-amplification with HER2, and was associated with a trend for poorer overall survival. Knockdown of GRB7 decreased proliferation and clonogenic survival, and induced apoptosis. Reverse phase protein array (RPPA) analyses revealed a role for PI3K, mammalian target of rapamycin (mTOR), MAPK, and receptor tyrosine kinase signalling in the oncogenic action of GRB7. Furthermore, the GRB7 and HER2 high-expressing OAC cell line Eso26 showed reduced cell proliferation upon GRB7 knockdown but was insensitive to HER2 inhibition by trastuzumab. Consistent with this, GRB7 knockdown in vivo with an inducible shRNA significantly inhibited tumour growth in cell line xenografts. HER2 expression did not predict sensitivity to trastuzumab, with Eso26 xenografts remaining refractory to trastuzumab treatment. Taken together, our study provides strong evidence for an oncogenic role for GRB7 in OAC and suggests that targeting GRB7 may be a potential therapeutic strategy for this cancer. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.
Asunto(s)
Adenocarcinoma/metabolismo , Biomarcadores de Tumor/metabolismo , Neoplasias Esofágicas/metabolismo , Proteína Adaptadora GRB7/metabolismo , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/mortalidad , Adenocarcinoma/patología , Animales , Antineoplásicos Inmunológicos/uso terapéutico , Western Blotting , Línea Celular Tumoral , Neoplasias Esofágicas/tratamiento farmacológico , Neoplasias Esofágicas/mortalidad , Neoplasias Esofágicas/patología , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Inmunohistoquímica , Ratones , Ratones Endogámicos NOD , Ratones SCID , Trasplante de Neoplasias , Pronóstico , Receptor ErbB-2/metabolismo , Análisis de Supervivencia , Trastuzumab/uso terapéuticoRESUMEN
Oncogenic mutations in the small GTPase Ras contribute to ~30% of human cancers. However, Ras mutations alone are insufficient for tumorigenesis, therefore it is paramount to identify cooperating cancer-relevant signaling pathways. We devised an in vivo near genome-wide, functional screen in Drosophila and discovered multiple novel, evolutionarily-conserved pathways controlling Ras-driven epithelial tumorigenesis. Human gene orthologs of the fly hits were significantly downregulated in thousands of primary tumors, revealing novel prognostic markers for human epithelial tumors. Of the top 100 candidate tumor suppressor genes, 80 were validated in secondary Drosophila assays, identifying many known cancer genes and multiple novel candidate genes that cooperate with Ras-driven tumorigenesis. Low expression of the confirmed hits significantly correlated with the KRASG12 mutation status and poor prognosis in pancreatic cancer. Among the novel top 80 candidate cancer genes, we mechanistically characterized the function of the top hit, the Tetraspanin family member Tsp29Fb, revealing that Tsp29Fb regulates EGFR signaling, epithelial architecture and restrains tumor growth and invasion. Our functional Drosophila screen uncovers multiple novel and evolutionarily conserved epithelial cancer genes, and experimentally confirmed Tsp29Fb as a key regulator of EGFR/Ras induced epithelial tumor growth and invasion.
Asunto(s)
Proteínas de Drosophila/genética , IMP Deshidrogenasa/genética , Neoplasias/genética , Tetraspanina 29/genética , Animales , Animales Modificados Genéticamente , Carcinogénesis/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Células Epiteliales/metabolismo , Células Epiteliales/patología , Femenino , Genes ras , Pruebas Genéticas/métodos , Humanos , IMP Deshidrogenasa/metabolismo , Masculino , Ratones , Neoplasias/metabolismo , Neoplasias/patología , Oncogenes , Transducción de Señal , Tetraspanina 29/metabolismo , Proteínas Supresoras de Tumor/genéticaRESUMEN
BACKGROUND: Triple negative breast cancer (TNBC) accounts for 16% of breast cancers and represents an aggressive subtype that lacks targeted therapeutic options. In this study, mass spectrometry (MS)-based tyrosine phosphorylation profiling identified aberrant FGFR3 activation in a subset of TNBC cell lines. This kinase was therefore evaluated as a potential therapeutic target. METHODS: MS-based tyrosine phosphorylation profiling was undertaken across a panel of 24 TNBC cell lines. Immunoprecipitation and Western blot were used to further characterize FGFR3 phosphorylation. Indirect immunofluorescence and confocal microscopy were used to determine FGFR3 localization. The selective FGFR1-3 inhibitor, PD173074 and siRNA knockdowns were used to characterize the functional role of FGFR3 in vitro. The TCGA and Metabric breast cancer datasets were interrogated to identify FGFR3 alterations and how they relate to breast cancer subtype and overall patient survival. RESULTS: High FGFR3 expression and phosphorylation were detected in SUM185PE cells, which harbor a FGFR3-TACC3 gene fusion. Low FGFR3 phosphorylation was detected in CAL51, MFM-223 and MDA-MB-231 cells. In SUM185PE cells, the FGFR3-TACC3 fusion protein contributed the majority of phosphorylated FGFR3, and largely localized to the cytoplasm and plasma membrane, with staining at the mitotic spindle in a small subset of cells. Knockdown of the FGFR3-TACC3 fusion and wildtype FGFR3 in SUM185PE cells decreased FRS2, AKT and ERK phosphorylation, and induced cell death. Knockdown of wildtype FGFR3 resulted in only a trend for decreased proliferation. PD173074 significantly decreased FRS2, AKT and ERK activation, and reduced SUM185PE cell proliferation. Cyclin A and pRb were also decreased in the presence of PD173074, while cleaved PARP was increased, indicating cell cycle arrest in G1 phase and apoptosis. Knockdown of FGFR3 in CAL51, MFM-223 and MDA-MB-231 cells had no significant effect on cell proliferation. Interrogation of public datasets revealed that increased FGFR3 expression in breast cancer was significantly associated with reduced overall survival, and that potentially oncogenic FGFR3 alterations (eg mutation and amplification) occur in the TNBC/basal, luminal A and luminal B subtypes, but are rare. CONCLUSIONS: These results indicate that targeting FGFR3 may represent a therapeutic option for TNBC, but only for patients with oncogenic FGFR3 alterations, such as the FGFR3-TACC3 fusion. Video abstract.
Asunto(s)
Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/metabolismo , Neoplasias de la Mama Triple Negativas/metabolismo , Línea Celular Tumoral , Proliferación Celular , Femenino , Puntos de Control de la Fase G1 del Ciclo Celular , Humanos , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Fosforilación , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/genética , Transducción de Señal , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/fisiopatologíaRESUMEN
Micro-RNAs (miRNAs) are potent regulators of gene expression and cellular phenotype. Each miRNA has the potential to target hundreds of transcripts within the cell thus controlling fundamental cellular processes such as survival and proliferation. Here, we exploit this important feature of miRNA networks to discover vulnerabilities in cancer phenotype, and map miRNA-target relationships across different cancer types. More specifically, we report the results of a functional genomics screen of 1280 miRNA mimics and inhibitors in eight cancer cell lines, and its presentation in a sophisticated interactive data portal. This resource represents the most comprehensive survey of miRNA function in oncology, incorporating breast cancer, prostate cancer and neuroblastoma. A user-friendly web portal couples this experimental data with multiple tools for miRNA target prediction, pathway enrichment analysis and visualization. In addition, the database integrates publicly available gene expression and perturbation data enabling tailored and context-specific analysis of miRNA function in a particular disease. As a proof-of-principle, we use the database and its innovative features to uncover novel determinants of the neuroblastoma malignant phenotype.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Predisposición Genética a la Enfermedad/genética , MicroARNs/genética , Neoplasias/genética , Línea Celular , Línea Celular Tumoral , Análisis por Conglomerados , Bases de Datos de Ácidos Nucleicos , Redes Reguladoras de Genes , Humanos , MicroARNs/clasificación , Neoplasias/patologíaRESUMEN
Dynamic assembly and disassembly of actin filaments is a major driving force for cell movements. Border cells in the Drosophila ovary provide a simple and genetically tractable model to study the mechanisms regulating cell migration. To identify new genes that regulate cell movement in vivo, we screened lethal mutations on chromosome 3R for defects in border cell migration and identified two alleles of the gene psidin (psid). In vitro, purified Psid protein bound F-actin and inhibited the interaction of tropomyosin with F-actin. In vivo, psid mutations exhibited genetic interactions with the genes encoding tropomyosin and cofilin. Border cells overexpressing Psid together with GFP-actin exhibited altered protrusion/retraction dynamics. Psid knockdown in cultured S2 cells reduced, and Psid overexpression enhanced, lamellipodial dynamics. Knockdown of the human homolog of Psid reduced the speed and directionality of migration in wounded MCF10A breast epithelial monolayers, whereas overexpression of the protein increased migration speed and altered protrusion dynamics in EGF-stimulated cells. These results indicate that Psid is an actin regulatory protein that plays a conserved role in protrusion dynamics and cell migration.
Asunto(s)
Proteínas Sanguíneas/metabolismo , Movimiento Celular , Extensiones de la Superficie Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Factores Despolimerizantes de la Actina/metabolismo , Animales , Proteínas Sanguíneas/química , Proteínas Sanguíneas/genética , Línea Celular Tumoral , Movimiento Celular/genética , Extensiones de la Superficie Celular/genética , Extensiones de la Superficie Celular/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Femenino , Regulación de la Expresión Génica , Silenciador del Gen , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Mutación , Ovario/citología , Tropomiosina/metabolismoRESUMEN
Hendra and Nipah viruses (genus Henipavirus, family Paramyxoviridae) are highly pathogenic bat-borne viruses. The need for high biocontainment when studying henipaviruses has hindered the development of therapeutics and knowledge of the viral infection cycle. We have performed a genome-wide siRNA screen at biosafety level 4 that identified 585 human proteins required for henipavirus infection. The host protein with the largest impact was fibrillarin, a nucleolar methyltransferase that was also required by measles, mumps and respiratory syncytial viruses for infection. While not required for cell entry, henipavirus RNA and protein syntheses were greatly impaired in cells lacking fibrillarin, indicating a crucial role in the RNA replication phase of infection. During infection, the Hendra virus matrix protein co-localized with fibrillarin in cell nucleoli, and co-associated as a complex in pulldown studies, while its nuclear import was unaffected in fibrillarin-depleted cells. Mutagenesis studies showed that the methyltransferase activity of fibrillarin was required for henipavirus infection, suggesting that this enzyme could be targeted therapeutically to combat henipavirus infections.
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Proteínas Cromosómicas no Histona/metabolismo , Infecciones por Henipavirus/virología , Virus Nipah/enzimología , Animales , Chlorocebus aethiops , Proteínas Cromosómicas no Histona/genética , Células HeLa , Virus Hendra/metabolismo , Humanos , Mutación , Virus Nipah/genética , Virus Nipah/patogenicidad , ARN Interferente Pequeño , Células Vero , Proteínas de la Matriz Viral/metabolismoRESUMEN
Hendra and Nipah viruses (family Paramyxoviridae, genus Henipavirus) are bat-borne viruses that cause fatal disease in humans and a range of other mammalian species. Gaining a deeper understanding of host pathways exploited by henipaviruses for infection may identify targets for new anti-viral therapies. Here we have performed genome-wide high-throughput agonist and antagonist screens at biosafety level 4 to identify host-encoded microRNAs (miRNAs) impacting henipavirus infection in human cells. Members of the miR-181 and miR-17~93 families strongly promoted Hendra virus infection. miR-181 also promoted Nipah virus infection, but did not affect infection by paramyxoviruses from other genera, indicating specificity in the virus-host interaction. Infection promotion was primarily mediated via the ability of miR-181 to significantly enhance henipavirus-induced membrane fusion. Cell signalling receptors of ephrins, namely EphA5 and EphA7, were identified as novel negative regulators of henipavirus fusion. The expression of these receptors, as well as EphB4, were suppressed by miR-181 overexpression, suggesting that simultaneous inhibition of several Ephs by the miRNA contributes to enhanced infection and fusion. Immune-responsive miR-181 levels was also up-regulated in the biofluids of ferrets and horses infected with Hendra virus, suggesting that the host innate immune response may promote henipavirus spread and exacerbate disease severity. This study is the first genome-wide screen of miRNAs influencing infection by a clinically significant mononegavirus and nominates select miRNAs as targets for future anti-viral therapy development.
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Infecciones por Henipavirus/genética , MicroARNs/genética , Internalización del Virus , Animales , Hurones , Técnica del Anticuerpo Fluorescente , Estudio de Asociación del Genoma Completo , Henipavirus , Secuenciación de Nucleótidos de Alto Rendimiento , Caballos , Humanos , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
The angiotensin type 1 receptor (AT1R) transactivates the epidermal growth factor receptor (EGFR) to mediate cellular growth, however, the molecular mechanisms involved have not yet been resolved. To address this, we performed a functional siRNA screen of the human kinome in human mammary epithelial cells that demonstrate a robust AT1R-EGFR transactivation. We identified a suite of genes encoding proteins that both positively and negatively regulate AT1R-EGFR transactivation. Many candidates are components of EGFR signalling networks, whereas others, including TRIO, BMX and CHKA, have not been previously linked to EGFR transactivation. Individual knockdown of TRIO, BMX or CHKA attenuated tyrosine phosphorylation of the EGFR by angiotensin II stimulation, but this did not occur following direct stimulation of the EGFR with EGF, indicating that these proteins function between the activated AT1R and the EGFR. Further investigation of TRIO and CHKA revealed that their activity is likely to be required for AT1R-EGFR transactivation. CHKA also mediated EGFR transactivation in response to another G protein-coupled receptor (GPCR) ligand, thrombin, indicating a pervasive role for CHKA in GPCR-EGFR crosstalk. Our study reveals the power of unbiased, functional genomic screens to identify new signalling mediators important for tissue remodelling in cardiovascular disease and cancer.
Asunto(s)
Células Epiteliales/metabolismo , Receptores ErbB/metabolismo , Glándulas Mamarias Humanas/metabolismo , Receptor de Angiotensina Tipo 1/metabolismo , Activación Transcripcional , Angiotensina II/metabolismo , Angiotensina II/farmacología , Línea Celular Transformada , Colina Quinasa/antagonistas & inhibidores , Colina Quinasa/genética , Colina Quinasa/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Receptores ErbB/genética , Femenino , Factores de Intercambio de Guanina Nucleótido/antagonistas & inhibidores , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Glándulas Mamarias Humanas/citología , Glándulas Mamarias Humanas/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptor de Angiotensina Tipo 1/genética , Transducción de Señal , Trombina/metabolismo , Trombina/farmacologíaRESUMEN
To provide a systematic analysis of genes that regulate epithelial cell migration, we performed a high throughput wound healing screen with MCF-10A breast epithelial cells, using siRNAs targeting 1,081 human genes encoding phosphatases, kinases and proteins predicted to influence cell migration and adhesion. The primary screen identified three categories of hits: those that accelerate, those that inhibit and those that impair migration with associated effects on cell proliferation or metabolism. Extensive validation of all the hits yielded 66 high confidence genes that, when downregulated, either accelerated or impaired migration; 42 of these high confidence genes have not been previously associated with motility or adhesion. Time-lapse video microscopy revealed a broad spectrum of phenotypic changes involving alterations in the extent and nature of disruption of cell-cell adhesion, directionality of motility, cell polarity and shape, and protrusion dynamics. Informatics analysis highlighted three major signalling nodes, beta-catenin, beta1-integrin and actin, and a large proportion of the genes that accelerated migration impaired cell-cell adhesion.
Asunto(s)
Movimiento Celular/genética , Células Epiteliales/citología , Células Epiteliales/metabolismo , Pruebas Genéticas , ARN Interferente Pequeño/genética , Actinas/genética , Actinas/metabolismo , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Línea Celular Tumoral , Proliferación Celular , Redes Reguladoras de Genes , Humanos , Integrina beta1/genética , Integrina beta1/metabolismo , Fenotipo , Reproducibilidad de los Resultados , Transducción de Señal , Factores de Tiempo , Transfección , Cicatrización de Heridas , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
Here we offer perspectives on phenotypic screening based on a wide-ranging discussion entitled "Phenotypic screening, target ID, and multi-omics: enabling more disease relevance in early discovery?" at the Screen Design and Assay Technology Special Interest Group Meeting at the 2023 SLAS Conference. During the session, the authors shared their own experience from within their respective organizations, followed by an open discussion with the audience. It was recognized that while substantial progress has been made towards translating disease-relevant phenotypic early discovery into clinical success, there remain significant operational and scientific challenges to implementing phenotypic screening efforts, and improving translation of screening hits comes with substantial resource demands and organizational commitment. This Perspective assesses progress, highlights pitfalls, and offers possible solutions to help unlock the therapeutic potential of phenotypic drug discovery. Areas explored comprise screening and hit validation strategy, choice of cellular model, moving beyond 2D cell culture into three dimensions, and leveraging high-dimensional data sets downstream of phenotypic screens.
Asunto(s)
Descubrimiento de Drogas , Opinión Pública , Descubrimiento de Drogas/métodos , FenotipoRESUMEN
The structures and functions of organelles in cells depend on each other but have not been systematically explored. We established stable knockout cell lines of peroxisomal, Golgi and endoplasmic reticulum genes identified in a whole-genome CRISPR knockout screen for inducers of mitochondrial biogenesis stress, showing that defects in peroxisome, Golgi and endoplasmic reticulum metabolism disrupt mitochondrial structure and function. Our quantitative total-organelle profiling approach for focussed ion beam scanning electron microscopy revealed in unprecedented detail that specific organelle dysfunctions precipitate multi-organelle biogenesis defects, impair mitochondrial morphology and reduce respiration. Multi-omics profiling showed a unified proteome response and global shifts in lipid and glycoprotein homeostasis that are elicited when organelle biogenesis is compromised, and that the resulting mitochondrial dysfunction can be rescued with precursors for ether-glycerophospholipid metabolic pathways. This work defines metabolic and morphological interactions between organelles and how their perturbation can cause disease.