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BACKGROUND: AP4 (TFAP4) encodes a basic helix-loop-helix leucine zipper (bHLH-LZ) transcription factor and is a direct target gene of the oncogenic transcription factor c-MYC. Here, we set out to determine the relevance of AP4 in human colorectal cancer (CRC) cells. METHODS: A CRISPR/Cas9 approach was employed to generate AP4-deficient CRC cell lines with inducible expression of c-MYC. Colony formation, ß-gal staining, immunofluorescence, comet and homologous recombination (HR) assays and RNA-Seq analysis were used to determine the effects of AP4 inactivation. qPCR and qChIP analyses was performed to validate differentially expressed AP4 targets. Expression data from CRC cohorts was subjected to bioinformatics analyses. Immunohistochemistry was used to evaluate AP4 targets in vivo. Ap4-deficient APCmin/+ mice were analyzed to determine conservation. Immunofluorescence, chromosome and micronuclei enumeration, MTT and colony formation assays were used to determine the effects of AP4 inactivation and target gene regulation on chromosomal instability (CIN) and drug sensitivity. RESULTS: Inactivation of AP4 in CRC cell lines resulted in increased spontaneous and c-MYC-induced DNA damage, chromosomal instability (CIN) and cellular senescence. AP4-deficient cells displayed increased expression of the long non-coding RNA MIR22HG, which encodes miR-22-3p and was directly repressed by AP4. Furthermore, Mediator of DNA damage Checkpoint 1 (MDC1), a central component of the DNA damage response and a known target of miR-22-3p, displayed decreased expression in AP4-deficient cells. Accordingly, MDC1 was directly induced by AP4 and indirectly by AP4-mediated repression of miR-22-3p. Adenomas and organoids from Ap4-deficient APCmin/+ mice displayed conservation of these regulations. Inhibition of miR-22-3p or ectopic MDC1 expression reversed the increased senescence, DNA damage, CIN and defective HR observed in AP4-deficient CRC cells. AP4-deficiency also sensitized CRC cells to 5-FU treatment, whereas ectopic AP4 conferred resistance to 5-FU in a miR-22-3p and MDC1-dependent manner. CONCLUSIONS: In summary, AP4, miR-22-3p and MDC1 form a conserved and coherent, regulatory feed-forward loop to promote DNA repair, which suppresses DNA damage, senescence and CIN, and contributes to 5-FU resistance. These findings explain how elevated AP4 expression contributes to development and chemo-resistance of colorectal cancer after c-MYC activation.
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Neoplasias Colorrectales , MicroARNs , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Proteínas de Ciclo Celular/genética , Inestabilidad Cromosómica , Neoplasias Colorrectales/genética , Daño del ADN , Fluorouracilo/farmacología , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , MicroARNs/genética , MicroARNs/metabolismo , Factores de Transcripción/genéticaRESUMEN
MicroRNAs (miRNAs) are important components of the signaling cascades that mediate and regulate tumor suppression exerted by p53. This review illustrates some of the main principles that underlie the mechanisms by which miRNAs participate in p53's function and how they were identified. Furthermore, the current status of the research on the connection between p53 and miRNAs, as well as alterations in the p53/miRNA pathways found in cancer will be summarized and discussed. In addition, experimental and bioinformatic approaches which can be applied to study the connection between p53 and miRNAs are described. Although, some of the central miRNA-encoding genes that mediate the effects of p53, such as the miR-34 and miR-200 families, have been identified, much more analyses remain to be performed to fully elucidate the connections between p53 and miRNAs.
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MicroARNs , Neoplasias , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Neoplasias/genética , Biología ComputacionalRESUMEN
BACKGROUND & AIMS: Combined inactivation of the microRNA 34a gene (MIR34A, by methylation) and the TP53 gene (by mutation or deletion) is observed in 50% of colorectal tumors that progress to distant metastases. We studied mice with intestinal disruption of Mir34a and Tp53 to investigate mechanisms of colorectal carcinogenesis and identify strategies to block these processes. METHODS: Mice with disruption of Mir34a and/or Tp53 specifically in intestinal epithelial cells (IECs) (Mir34aΔIEC mice, Tp53ΔIEC mice, and Mir34aΔIEC/Tp53ΔIEC mice) and controls (Mir34aFl/Fl/Tp53Fl/Fl) were given azoxymethane to induce colorectal carcinogenesis. Some mice were given intraperitoneal injections of an antibody against mouse interleukin 6 receptor (IL6R), or received an inhibitor of PAI1 (tiplaxtinin) in their chow. Intestinal tissues were collected and analyzed by immunohistochemistry; gene expression profiles were analyzed by RNA sequencing. We determined the expression and localization of PAI1 in 61 human primary colon cancers and compared them to MIR34A methylation and inactivating mutations in TP53. Data on mRNA levels, methylation, and clinical features of 628 colon and rectal adenocarcinomas were obtained from The Cancer Genome Atlas portal. RESULTS: Mir34aΔIEC/Tp53ΔIEC mice developed larger and more colorectal tumors, with increased invasion of surrounding tissue and metastasis to lymph nodes, than control mice or mice with disruption of either gene alone. Cells in tumors from the Mir34aΔIEC/Tp53ΔIEC mice had decreased apoptosis and increased proliferation compared to tumor cells from control mice, and expressed higher levels of genes, that regulate inflammation (including Il6r and Stat3) and epithelial-mesenchymal transition. The gene expression pattern of the tumors from Mir34aΔIEC/Tp53ΔIEC mice was similar to that of human colorectal tumor consensus molecular subtype 4 (mesenchymal, invasive). We identified the Pai1 messenger RNA as a target of Mir34a; levels of PAI1 protein were increased in primary colon cancer samples, that displayed methylation of MIR34A and mutational inactivation of TP53. Administration of tiplaxtinin or anti-IL6R antibody to Mir34aΔIEC/Tp53ΔIEC mice decreased proliferation of cancer cells, and reduced colorectal tumor invasion and metastasis. CONCLUSIONS: In mice, we demonstrated that combined inactivation of Mir34a and Tp53 promotes azoxymethane-induced colorectal carcinogenesis and tumor progression and metastasis by increasing levels of IL6R and PAI1. Strategies to inhibit these processes might be developed to slow progression of colorectal cancer.
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Neoplasias Colorrectales/genética , Silenciador del Gen , Genes p53 , MicroARNs/genética , Receptores de Interleucina-6/metabolismo , Serpina E2/metabolismo , Animales , Azoximetano , Carcinógenos , Neoplasias Colorrectales/inducido químicamente , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Progresión de la Enfermedad , Células Epiteliales/fisiología , Mucosa Intestinal/citología , RatonesRESUMEN
We determined the effect of p53 activation on de novo protein synthesis using quantitative proteomics (pulsed stable isotope labeling with amino acids in cell culture/pSILAC) in the colorectal cancer cell line SW480. This was combined with mRNA and noncoding RNA expression analyses by next generation sequencing (RNA-, miR-Seq). Furthermore, genome-wide DNA binding of p53 was analyzed by chromatin-immunoprecipitation (ChIP-Seq). Thereby, we identified differentially regulated proteins (542 up, 569 down), mRNAs (1258 up, 415 down), miRNAs (111 up, 95 down) and lncRNAs (270 up, 123 down). Changes in protein and mRNA expression levels showed a positive correlation (r = 0.50, p < 0.0001). In total, we detected 133 direct p53 target genes that were differentially expressed and displayed p53 occupancy in the vicinity of their promoter. More transcriptionally induced genes displayed occupied p53 binding sites (4.3% mRNAs, 7.2% miRNAs, 6.3% lncRNAs, 5.9% proteins) than repressed genes (2.4% mRNAs, 3.2% miRNAs, 0.8% lncRNAs, 1.9% proteins), suggesting indirect mechanisms of repression. Around 50% of the down-regulated proteins displayed seed-matching sequences of p53-induced miRNAs in the corresponding 3'-UTRs. Moreover, proteins repressed by p53 significantly overlapped with those previously shown to be repressed by miR-34a. We confirmed up-regulation of the novel direct p53 target genes LINC01021, MDFI, ST14 and miR-486 and showed that ectopic LINC01021 expression inhibits proliferation in SW480 cells. Furthermore, KLF12, HMGB1 and CIT mRNAs were confirmed as direct targets of the p53-induced miR-34a, miR-205 and miR-486-5p, respectively. In line with the loss of p53 function during tumor progression, elevated expression of KLF12, HMGB1 and CIT was detected in advanced stages of cancer. In conclusion, the integration of multiple omics methods allowed the comprehensive identification of direct and indirect effectors of p53 that provide new insights and leads into the mechanisms of p53-mediated tumor suppression.
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MicroARNs/metabolismo , ARN Largo no Codificante/metabolismo , ARN Mensajero/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Arginina , Isótopos de Carbono , Línea Celular Tumoral , ADN/metabolismo , Humanos , Marcaje Isotópico , Lisina , Isótopos de Nitrógeno , Análisis de Secuencia de ADN , Proteína p53 Supresora de Tumor/genéticaRESUMEN
The epithelial-mesenchymal-transition (EMT) represents a morphogenetic program involved in developmental processes such as gastrulation and neural crest formation. The EMT program is co-opted by epithelial tumor cells and endows them with features necessary for spreading to distant sites, such as invasion, migration, apoptosis resistance and stemness. Thereby, EMT facilitates metastasis formation and therapy resistance. A growing number of transcription factors has been implicated in the regulation of EMT. These include EMT-inducing transcription factors (EMT-TFs), the most prominent being SNAIL, SLUG, ZEB1, ZEB2 and TWIST, and negative regulators of EMT, such as p53. Furthermore, a growing number of microRNAs, such as members of the miR-200 and miR-34 family, have been characterized as negative regulators of EMT. EMT-TFs and microRNAs, such as ZEB1/2 and miR-200 or SNAIL and miR-34, are often engaged in double-negative feedback loops forming bistable switches controlling the transitions from epithelial to the mesenchymal cell states. Within this chapter, we will provide a comprehensive overview over the transcription factors and microRNAs that have been implicated in the regulation of EMT in colorectal cancer. Furthermore, we will highlight the regulatory connections between EMT-TFs and miRNAs to illustrate common principles of their interaction that regulate EMTs.
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Neoplasias Colorrectales/genética , Transición Epitelial-Mesenquimal/genética , Regulación Neoplásica de la Expresión Génica/genética , MicroARNs/genética , Factores de Transcripción/genética , Neoplasias Colorrectales/etiología , Neoplasias Colorrectales/patología , Retroalimentación Fisiológica , Humanos , Modelos Biológicos , Proteínas de Neoplasias/fisiologíaRESUMEN
BACKGROUND: Loss of the p53-inducible LINC01021 in p53-proficient CRC cell lines results in increased sensitivity to DNA-damaging chemotherapeutics. Here, we comprehensively analyze how LINC01021 affects the p53-induced transcriptional program. METHODS: Using a CRISPR/Cas9-approach, we deleted the p53 binding site in the LINC01021 promoter of SW480 colorectal cancer cells and subjected them to RNA-Seq analysis after the activation of ectopic p53. RNA affinity purification followed by mass spectrometry was used to identify proteins associated with LINC01021. RESULTS: Loss of the p53-inducibility of LINC01021 resulted in an ~1.8-fold increase in the number of significantly regulated mRNAs compared to LINC01021 wild-type cells after ectopic activation of p53. A subset of direct p53 target genes, such as NOXA and FAS, displayed significantly stronger induction when the p53-inducibility of LINC01021 was abrogated. Loss of the p53-inducibility of LINC01021 resulted in alternative splicing of a small number of mRNAs, such as ARHGAP12, HSF2, and LYN. Several RNA binding proteins involved in pre-mRNA splicing were identified as interaction partners of LINC01021 by mass spectrometry. CONCLUSIONS: Our results suggest that LINC01021 may restrict the extent and strength of p53-mediated transcriptional changes via context-dependent regulation of the expression and splicing of a subset of p53-regulated genes.
RESUMEN
The gene encoding the miR-34a microRNA is a transcriptional target of the p53 tumor suppressor protein and subject to epigenetic inactivation in colorectal cancer and numerous other tumor types. Here, we combined pulsed SILAC (pSILAC) and microarray analyses to identify miR-34a-induced changes in protein and mRNA expression. pSILAC allowed to quantify the de novo protein synthesis of 1206 proteins after activation of a conditional miR-34a allele in a colorectal cancer cell line. â¼19% of the detected proteins were differentially regulated, with 113 proteins being down- and 115 up-regulated. The proteins with a miR-34a seed-matching-sequence in the 3'-untranslated region (UTR) of the corresponding mRNA showed a clear bias toward translational repression. Proteins involved in DNA replication, e.g. the MCM proteins, and cell proliferation, were over-represented among indirectly down-regulated proteins lacking a miR-34a seed-match. The decrease in de novo protein synthesis of direct miR-34a targets correlated with reduced levels of the corresponding mRNA in most cases, indicating an interdependence of both types of regulation. In addition, 43 mRNAs encoding proteins not detected by pSILAC were down-regulated after miR-34a expression and contained miR-34a seed-matches. The direct regulation of selected miR-34a target-mRNAs was confirmed using reporter assays. Via down-regulation of the proteins encoded by these mRNAs miR-34a presumably inhibits glycolysis (LDHA), WNT-signaling (LEF1), invasion/migration (AXL) and lipid metabolism (ACSL1, ACSL4). Furthermore, miR-34a may activate p53 by inhibiting its acetylation (MTA2, HDAC1) and degradation (YY1). In summary, miR-34a presumably participates in multiple tumor suppressive pathways by directly and indirectly suppressing the expression of numerous, critical proteins.
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Expresión Génica , Genoma Humano , MicroARNs/fisiología , Proteoma/genética , ARN Mensajero/metabolismo , Línea Celular Tumoral , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Genes Reporteros , Genes Supresores de Tumor , Humanos , Marcaje Isotópico , Luciferasas de Renilla/biosíntesis , Luciferasas de Renilla/genética , Redes y Vías Metabólicas/genética , MicroARNs/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteoma/metabolismo , ARN Mensajero/genéticaRESUMEN
In the recent years, microRNAs (miRNAs) were identified as important components of the signaling cascades that mediate and regulate tumor suppression exerted by p53. This review illustrates some of the main principles that underlie the mechanisms by which miRNAs participate in p53's function and how they were identified. Furthermore, the current status of the research on the connection between p53 and miRNAs, as well as alterations in the p53/miRNA pathways found in cancer will be summarized and discussed. In addition, experimental and bioinformatics approaches, which can be applied to study the connection between p53 and miRNAs are described. Although, some of the central miRNA-encoding genes that mediate the effects of p53, such as the miR-34 and miR-200 families, have been identified, many additional analyses remain to be performed to fully elucidate the connections between p53 and miRNAs.
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Biología Computacional , Redes Reguladoras de Genes/genética , MicroARNs/metabolismo , Neoplasias/genética , Proteína p53 Supresora de Tumor/metabolismo , Animales , Regulación Neoplásica de la Expresión Génica , Humanos , MicroARNs/genética , Proteína p53 Supresora de Tumor/genéticaRESUMEN
BACKGROUND: The deregulated expression of the c-MYC oncogene activates p53, which is presumably mediated by ARF/INK4, as well as replication-stress-induced DNA damage. Here, we aimed to determine whether the c-MYC-inducible AP4 transcription factor plays a role in this context using a genetic approach. METHODS: We used a CRISPR/Cas9 approach to generate AP4- and/or p53-deficient derivatives of MCF-7 breast cancer cells harboring an ectopic, inducible c-MYC allele. Cell proliferation, senescence, DNA damage, and comprehensive RNA expression profiles were determined after activation of c-MYC. In addition, we analyzed the expression data from primary breast cancer samples. RESULTS: Loss of AP4 resulted in elevated levels of both spontaneous and c-MYC-induced DNA damage, senescence, and diminished cell proliferation. Deletion of p53 in AP4-deficient cells reverted senescence and proliferation defects without affecting DNA damage levels. RNA-Seq analyses showed that loss of AP4 enhanced repression of DREAM and E2F target genes after p53 activation by c-MYC. Depletion of p21 or the DREAM complex component LIN37 abrogated this effect. These p53-dependent effects were conserved on the level of clinical and gene expression associations found in primary breast cancer tumors. CONCLUSIONS: Our results establish AP4 as a pivotal factor at the crossroads of c-MYC, E2F, and p53 target gene regulation.
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The miR-34a and miR-34b/c encoding genes represent direct targets of the p53 transcription factor, and presumably mediate part of the tumor suppressive effects of p53. Here, we sought to determine their functional relevance by inactivating miR-34a and/or miR-34b/c using a CRISPR/Cas9 approach in the colorectal cancer (CRC) cell line HCT116. Concomitant deletion of miR-34a and miR-34b/c resulted in significantly reduced suppression of proliferation after p53 activation, enhanced migration, invasion and EMT, as well as reduced sensitivity to chemotherapeutics, increased stress-induced autophagic flux, decreased apoptosis and upregulation of autophagy-related genes after 5-FU treatment. However, inactivation of singular miR-34a or miR-34b/c had little effects on the aforementioned processes. RNA-Seq analysis revealed that concomitant deletion of miR-34a/b/c caused EMT signature enrichment, impaired gene repression by the p53-DREAM pathway and elevated autophagy after 5-FU treatment. A gene signature comprised of mRNAs significantly upregulated after combined inactivation of miR-34a and miR-34b/c showed a significant association with the invasive colon cancer subtype CMS4 and poor overall survival in two CRC patient cohorts, and with 5-FU resistance in CRC cell lines. In miR-34a/b/c-deficient cells the upregulated miR-34 target FOXM1 directly induced p62 and ATG9A, which increased autophagy and consequently attenuated apoptosis and rendered the miR-34a/b/c-KO cells more resistant to 5-FU. Inhibition of autophagy by depletion of ATG9A or chloroquine re-sensitized miR-34a/b/c-deficient HCT116 cells to 5-FU. In summary, our findings show a complementary role of miR-34a and miR-34b/c in the regulation of EMT and autophagy which may be relevant for CRC therapy in the future.
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Neoplasias Colorrectales , MicroARNs , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Células HCT116 , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Sistemas CRISPR-Cas/genética , Línea Celular Tumoral , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Fluorouracilo/farmacología , Autofagia/genética , Regulación Neoplásica de la Expresión GénicaRESUMEN
The CSF1 receptor (CSF1R) encoding mRNA represents a direct target of miR-34a. However, the in vivo relevance of the suppression of CSF1R by miR-34a for intestinal tumor suppression mediated by the p53/miR-34a pathway has remained unknown. Here, Apc Min/+ mice with intestinal-epithelial cell (IEC)-specific deletions of Mir34a showed increased formation of adenomas and decreased survival, whereas deletion of Csf1r decreased adenoma formation and increased survival. In adenomas deletion of Mir34a enhanced proliferation, STAT3 signaling, infiltration with fibroblasts, immune cells and microbes, and tumor stem cell abundance and decreased apoptosis. Deletion of Csf1r had the opposite effects. In addition, homeostasis of intestinal secretory and stem cells, and tumoroid formation were affected in opposite directions by deletion of Mir34a and CSF1R. Concomitant deletion of Csf1r and Mir34a neutralized the effects of the single deletions. mRNAs containing Mir34a seed-matching sites, which encode proteins related to EMT (epithelial-mesenchymal transition), stemness and Wnt signaling, were enriched after Mir34a inactivation in adenomas and derived tumoroids. Netrin-1/Ntn1 and Transgelin/Tagln were characterized as direct targets of Mir34a and Csf1r signaling. Mir34a-inactivation related expression signatures were associated with CMS4/CRISB+D, stage 4 CRCs and poor patient survival. In tumoroids the loss of Mir34a conferred resistance to 5-FU which was mediated by Csf1r. This study provides genetic evidence for a requirement of Mir34a-mediated Csf1r suppression for intestinal stem/secretory cell homeostasis and tumor suppression, and suggests that therapeutic targeting of CSF1R may be effective for the treatment of CRCs with defects in the p53/miR-34a pathway.
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Adenoma , MicroARNs , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos , Adenoma/genética , Animales , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Fluorouracilo , Regulación Neoplásica de la Expresión Génica/genética , Ratones , MicroARNs/genética , MicroARNs/metabolismo , Netrina-1/genética , Netrina-1/metabolismo , ARN Mensajero , Proteínas Tirosina Quinasas Receptoras/genética , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Resistance towards cancer treatment represents a major clinical obstacle, preventing cure of cancer patients. To gain mechanistic insights, we developed a model for acquired resistance to chemotherapy by treating mice carrying patient derived xenografts (PDX) of acute lymphoblastic leukemia with widely-used cytotoxic drugs for 18 consecutive weeks. In two distinct PDX samples, tumors initially responded to treatment, until stable disease and eventually tumor re-growth evolved under therapy, at highly similar kinetics between replicate mice. Notably, replicate tumors developed different mutations in TP53 and individual sets of chromosomal alterations, suggesting independent parallel clonal evolution rather than selection, driven by a combination of stochastic and deterministic processes. Transcriptome and proteome showed shared dysregulations between replicate tumors providing putative targets to overcome resistance. In vivo CRISPR/Cas9 dropout screens in PDX revealed broad dependency on BCL2, BRIP1 and COPS2. Accordingly, venetoclax re-sensitized derivative tumors towards chemotherapy, despite genomic heterogeneity, demonstrating direct translatability of the approach. Hence, despite the presence of multiple resistance-associated genomic alterations, effective rescue treatment for polychemotherapy-resistant tumors can be identified using functional testing in preclinical models.
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Antineoplásicos , Neoplasias , Humanos , Ratones , Animales , Sistemas CRISPR-Cas , Antineoplásicos/uso terapéutico , Neoplasias/genética , Modelos Animales de Enfermedad , Transcriptoma , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The c-MYC oncogene is activated in ~50% of all tumors and its product, the c-MYC transcription factor, regulates numerous processes, which contribute to tumor initiation and progression. Therefore, the genome-wide characterization of c-MYC targets and their role in different tumor entities is a recurrent theme in cancer research. Recently, next-generation sequencing (NGS) has become a powerful tool to analyze mRNA and miRNA expression, as well as DNA binding of proteins in a genome-wide manner with an extremely high resolution and coverage. Since the c-MYC transcription factor regulates mRNA and miRNA expression by binding to specific DNA elements in the vicinity of promoters, NGS can be used to generate integrated representations of c-MYC-mediated regulations of gene transcription and chromatin modifications. Here, we provide protocols and examples of NGS-based analyses of c-MYC-regulated mRNA and miRNA expression, as well as of DNA binding by c-MYC. Furthermore, we describe the validation of single c-MYC targets identified by NGS . Taken together, these approaches allow an accelerated and comprehensive analysis of c-MYC function in numerous cellular contexts. Ultimately, these analyses will further illuminate the role of this important oncogene.
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Estudio de Asociación del Genoma Completo/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Proteínas Proto-Oncogénicas c-myc/genética , ADN/genética , Proteínas de Unión al ADN/genética , Expresión Génica , Perfilación de la Expresión Génica/métodos , Genes myc , Humanos , MicroARNs/genética , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN/genética , ARN Mensajero/genética , Análisis de Secuencia de ADN/métodosRESUMEN
Two contributors' names were missing in the original version of the authorship of the paper [...].
RESUMEN
BACKGROUND & AIMS: The miR-34a gene is a direct target of p53 and is commonly silenced in colorectal cancer (CRC). Here we identified the receptor tyrosine kinase CSF1R as a direct miR-34a target and characterized CSF1R as an effector of p53/miR-34a-mediated CRC suppression. METHODS: Analyses of TCGA-COAD and three other CRC cohorts for association of mRNA expression and signatures with patient survival and molecular subtypes. Bioinformatics identification and experimental validation of miRNA and transcription factor targets. Functional analysis of factors/pathways in the regulation of epithelial-mesenchymal transition (EMT), invasion, migration, acquired chemo-resistance and metastasis. Analyses of protein expression and CpG methylation within primary human colon cancer samples. RESULTS: In primary CRCs increased CSF1R, CSF1 and IL34 expression was associated with poor patient survival and a mesenchymal-like subtype. CSF1R displayed an inverse correlation with miR-34a expression. This was explained by direct inhibition of CSF1R by miR-34a. Furthermore, p53 repressed CSF1R via inducing miR-34a, whereas SNAIL induced CSF1R both directly and indirectly via repressing miR-34a in a coherent feed-forward loop. Activation of CSF1R induced EMT, migration, invasion and metastasis of CRC cells via STAT3-mediated down-regulation of miR-34a. 5-FU resistance of CRC cells was mediated by CpG-methylation of miR-34a and the resulting elevated expression of CSF1R. In primary CRCs elevated expression of CSF1R was detected at the tumor invasion front and was associated with CpG methylation of the miR-34a promoter as well as distant metastasis. CONCLUSIONS: The reciprocal inhibition between miR-34a and CSF1R and its loss in tumor cells may be relevant for therapeutic and prognostic approaches towards CRC management.
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Neoplasias Colorrectales/genética , Regulación Neoplásica de la Expresión Génica , MicroARNs/metabolismo , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Línea Celular Tumoral , Colectomía , Colon/patología , Colon/cirugía , Neoplasias Colorrectales/mortalidad , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/terapia , Islas de CpG/genética , Metilación de ADN , Resistencia a Antineoplásicos/genética , Epigénesis Genética , Transición Epitelial-Mesenquimal/genética , Retroalimentación Fisiológica , Femenino , Fluorouracilo/farmacología , Fluorouracilo/uso terapéutico , Humanos , Estimación de Kaplan-Meier , Masculino , MicroARNs/genética , Proctectomía , Pronóstico , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Recto/patología , Recto/cirugía , Factor de Transcripción STAT3/metabolismo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Aurora kinases are highly conserved proteins with important roles in mitosis. Metazoans contain two kinases, Aurora A and B, which contribute distinct functions at the spindle poles and the equatorial region respectively. It is not currently known whether the specialized functions of the two kinases arose after their duplication in animal cells or were already present in their ancestral kinase. We show that Dictyostelium discoideum contains a single Aurora kinase, DdAurora, that displays characteristics of both Aurora A and B. Like Aurora A, DdAurora has an extended N-terminal domain with an A-box sequence and localizes at the spindle poles during early mitosis. Like Aurora B, DdAurora binds to its partner DdINCENP and localizes on centromeres at metaphase, the central spindle during anaphase, and the cleavage furrow at the end of cytokinesis. DdAurora also has several unusual properties. DdAurora remains associated with centromeres in anaphase, and this association does not require an interaction with DdINCENP. DdAurora then localizes at the cleavage furrow, but only at the end of cytokinesis. This localization is dependent on DdINCENP and the motor proteins Kif12 and myosin II. Thus, DdAurora may represent the ancestral kinase that gave rise to the different Aurora kinases in animals and also those in other organisms.
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Dictyostelium/enzimología , Proteínas Serina-Treonina Quinasas/metabolismo , Secuencia de Aminoácidos , Anafase , Animales , Aurora Quinasa B , Aurora Quinasas , Centrómero/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Dictyostelium/citología , Dictyostelium/genética , Dictyostelium/fisiología , Evolución Molecular , Humanos , Proteínas Motoras Moleculares/metabolismo , Datos de Secuencia Molecular , Filogenia , Plantas/genética , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Estructura Terciaria de Proteína , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Saccharomyces cerevisiae/genética , Alineación de Secuencia , Huso Acromático/metabolismo , Xenopus laevis/genéticaRESUMEN
The gene encoding the transcription factor TFAP4/AP4 represents a direct target of the c-MYC oncoprotein. Here, we deleted Ap4 in ApcMin mice, a preclinical model of inherited colorectal cancer. Ap4 deficiency extends their average survival by 110 days and decreases the formation of intestinal adenomas and tumor-derived organoids. The effects of Ap4 deletion are presumably due to the reduced number of functional intestinal stem cells (ISCs) amenable to adenoma-initiating mutational events. Deletion of Ap4 also decreases the number of colonic stem cells and increases the number of Paneth cells. Expression profiling revealed that ISC signatures, as well as the Wnt/ß-catenin and Notch signaling pathways are downregulated in Ap4-deficient adenomas and intestinal organoids. AP4-associated signatures are conserved between murine adenomas and human colorectal cancer samples. Our results establish Ap4 as rate-limiting mediator of adenoma initiation, as well as regulator of intestinal and colonic stem cell and Paneth cell homeostasis.
Asunto(s)
Adenoma/genética , Colon/metabolismo , Neoplasias Colorrectales/genética , Regulación Neoplásica de la Expresión Génica , Organoides/metabolismo , Células Madre/metabolismo , Factores de Transcripción/genética , Adenoma/metabolismo , Proteína de la Poliposis Adenomatosa del Colon/genética , Animales , Colon/patología , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Simulación por Computador , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Homeostasis , Humanos , Mucosa Intestinal/metabolismo , Neoplasias Intestinales/metabolismo , Neoplasias Intestinales/patología , Intestinos/patología , Ratones , Ratones Noqueados , Organoides/patología , Células de Paneth/patología , Receptores Notch/metabolismo , Células Madre/citología , Vía de Señalización WntRESUMEN
Purpose: Colorectal cancers are composed of phenotypically different tumor cell subpopulations within the same core genetic background. Here, we identify high expression of the TALE transcription factor PBX3 in tumor cells undergoing epithelial-mesenchymal transition (EMT), analyze PBX3 regulation, and determine clinical associations in colorectal cancer.Experimental design: We used transcriptomic and in situ analyses to identify PBX3 expression in colorectal cancer and cell biology approaches to determine its regulation and function. Clinical associations were analyzed in independent tissue collections and gene expression datasets of colorectal cancers with recorded follow-up data.Results: PBX3 was expressed in tumor cells with high WNT activity undergoing EMT at the leading tumor edge of colorectal cancers, whereas stromal cells were PBX3 negative. PBX3 expression was induced by WNT activation and by the EMT transcription factors SNAIL and ZEB1, whereas these effects were mediated indirectly through microRNA miR-200. PBX3 was required for a full EMT phenotype in colon cancer cells. On the protein level, PBX3 expression indicated poor cancer-specific and disease-free survival in a cohort of 244 UICC stage II colorectal cancers, and was associated with metastasis in a case-control collection consisting of 90 cases with or without distant metastasis. On the mRNA level, high PBX3 expression was strongly linked to poor disease-free survival.Conclusions: PBX3 is a novel indicator of EMT in colorectal cancer, part of an EMT regulatory network, and a promising prognostic predictor that may aid in therapeutic decision making for patients with colorectal cancer. Clin Cancer Res; 24(8); 1974-86. ©2018 AACR.
Asunto(s)
Neoplasias Colorrectales/genética , Neoplasias Colorrectales/mortalidad , Transición Epitelial-Mesenquimal/genética , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Proteínas de Homeodominio/genética , Proteínas Proto-Oncogénicas/genética , Línea Celular Tumoral , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Progresión de la Enfermedad , Proteínas de Homeodominio/metabolismo , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Pronóstico , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal , Proteínas Wnt/metabolismoRESUMEN
We have identified a DNA methyltransferase of the Dnmt2 family in Dictyostelium that was denominated DnmA. Expression of the dnmA gene is downregulated during the developmental cycle. Overall DNA methylation in Dictyostelium is approximately 0.2% of the cytosine residues, which indicates its restriction to a limited set of genomic loci. Bisulfite sequencing of specific sites revealed that DnmA is responsible for methylation of mostly asymmetric C-residues in the retrotransposons DIRS-1 and Skipper. Disruption of the gene resulted in a loss of methylation and in increased transcription and mobilization of Skipper. Skipper transcription was also upregulated in strains that had genes encoding components of the RNA interference pathway disrupted. In contrast, DIRS-1 expression was not affected by a loss of DnmA but was strongly increased in strains that had the RNA-directed RNA polymerase gene rrpC disrupted. A large number of siRNAs were found that corresponded to the DIRS-1 sequence, suggesting concerted regulation of DIRS-1 expression by RNAi and DNA modification. No siRNAs corresponding to the standard Skipper element were found. The data show that DNA methylation plays a crucial role in epigenetic gene silencing in Dictyostelium but that different, partially overlapping mechanisms control transposon silencing.