ABSTRACT
BACKGROUND: Non-coding RNAs represent a large part of the human transcriptome and have been shown to play an important role in disease such as cancer. However, their biological functions are still incompletely understood. Among non-coding RNAs, circular RNAs (circRNAs) have recently been identified for their microRNA (miRNA) sponge function which allows them to modulate the expression of miRNA target genes by taking on the role of competitive endogenous RNAs (ce-circRNAs). Today, most computational tools are not adapted to the search for ce-circRNAs or have not been developed for the search for ce-circRNAs from user's transcriptomic data. RESULTS: In this study, we present Cirscan (CIRcular RNA Sponge CANdidates), an interactive Shiny application that automatically infers circRNA-miRNA-mRNA networks from human multi-level transcript expression data from two biological conditions (e.g. tumor versus normal conditions in the case of cancer study) in order to identify on a large scale, potential sponge mechanisms active in a specific condition. Cirscan ranks each circRNA-miRNA-mRNA subnetwork according to a sponge score that integrates multiple criteria based on interaction reliability and expression level. Finally, the top ranked sponge mechanisms can be visualized as networks and an enrichment analysis is performed to help its biological interpretation. We showed on two real case studies that Cirscan is capable of retrieving sponge mechanisms previously described, as well as identifying potential novel circRNA sponge candidates. CONCLUSIONS: Cirscan can be considered as a companion tool for biologists, facilitating their ability to prioritize sponge mechanisms for experimental validations and identifying potential therapeutic targets. Cirscan is implemented in R, released under the license GPL-3 and accessible on GitLab ( https://gitlab.com/geobioinfo/cirscan_Rshiny ). The scripts used in this paper are also provided on Gitlab ( https://gitlab.com/geobioinfo/cirscan_paper ).
Subject(s)
MicroRNAs , Neoplasms , Humans , MicroRNAs/genetics , MicroRNAs/metabolism , RNA, Circular/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reproducibility of Results , Gene Regulatory NetworksABSTRACT
The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that has been shown to be an essential regulator of a broad spectrum of biological activities required for maintaining the body's vital functions. AhR also plays a critical role in tumorigenesis. Its role in cancer is complex, encompassing both pro- and anti-tumorigenic activities. Its level of expression and activity are specific to each tumor and patient, increasing the difficulty of understanding the activating or inhibiting roles of AhR ligands. We explored the role of AhR in tumor cell lines and patients using genomic data sets and discuss the extent to which AhR can be considered as a therapeutic target.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Disease Susceptibility , Neoplasms/etiology , Neoplasms/metabolism , Receptors, Aryl Hydrocarbon/genetics , Receptors, Aryl Hydrocarbon/metabolism , Animals , Biomarkers , Cell Line, Tumor , Gene Expression Profiling , Gene Expression Regulation , Humans , Meta-Analysis as Topic , Mutation , Neoplasms/pathology , Oncogenes , Precision Medicine , Transcriptome , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolismABSTRACT
OBJECTIVE: Helicobacter pylori (Hp) is a major risk factor for gastric cancer (GC). Hp promotes DNA damage and proteasomal degradation of p53, the guardian of genome stability. Hp reduces the expression of the transcription factor USF1 shown to stabilise p53 in response to genotoxic stress. We investigated whether Hp-mediated USF1 deregulation impacts p53-response and consequently genetic instability. We also explored in vivo the role of USF1 in gastric carcinogenesis. DESIGN: Human gastric epithelial cell lines were infected with Hp7.13, exposed or not to a DNA-damaging agent camptothecin (CPT), to mimic a genetic instability context. We quantified the expression of USF1, p53 and their target genes, we determined their subcellular localisation by immunofluorescence and examined USF1/p53 interaction. Usf1-/- and INS-GAS mice were used to strengthen the findings in vivo and patient data examined for clinical relevance. RESULTS: In vivo we revealed the dominant role of USF1 in protecting gastric cells against Hp-induced carcinogenesis and its impact on p53 levels. In vitro, Hp delocalises USF1 into foci close to cell membranes. Hp prevents USF1/p53 nuclear built up and relocates these complexes in the cytoplasm, thereby impairing their transcriptional function. Hp also inhibits CPT-induced USF1/p53 nuclear complexes, exacerbating CPT-dependent DNA damaging effects. CONCLUSION: Our data reveal that the depletion of USF1 and its de-localisation in the vicinity of cell membranes are essential events associated to the genotoxic activity of Hp infection, thus promoting gastric carcinogenesis. These findings are also of clinical relevance, supporting USF1 expression as a potential marker of GC susceptibility.
Subject(s)
Carcinogenesis , Gastric Mucosa , Helicobacter Infections/metabolism , Helicobacter pylori , Stomach Neoplasms , Tumor Suppressor Protein p53/genetics , Upstream Stimulatory Factors/metabolism , Animals , Carcinogenesis/genetics , Carcinogenesis/metabolism , Cell Line , DNA Damage , Gastric Mucosa/metabolism , Gastric Mucosa/microbiology , Gastric Mucosa/pathology , Genomic Instability , Helicobacter pylori/metabolism , Helicobacter pylori/pathogenicity , Humans , Mice , Proteasome Endopeptidase Complex/metabolism , Stomach Neoplasms/genetics , Stomach Neoplasms/metabolism , Stomach Neoplasms/microbiology , UbiquitinationABSTRACT
BACKGROUND: With the development of precision oncology, Molecular Tumor Boards (MTB) are developing in many institutions. However, the implementation of MTB in routine clinical practice has still not been thoroughly studied. MATERIAL AND METHODS: Since the first drugs approved for targeted therapies, patient tumor samples were centralized to genomic testing platforms. In our institution, all tumor samples have been analyzed since 2014 by Next Generation Sequencing (NGS). In 2015, we established a regional MTB to discuss patient cases with 1 or more alterations identified by NGS, in genes different from those related to drug approval. We conducted a retrospective comparative analysis to study whether our MTB increased the prescriptions of Molecular Targeted Therapies (MTT) and the inclusions of patients in clinical trials with MTT, in comparison with patients with available NGS data but no MTB discussion. RESULTS: In 2014, 86 patients had UGA, but the results were not available to clinicians and not discussed in MTB. During the years 2015 and 2016, 113 patients with an UGA (unreferenced genomic alteration) were discussed in MTB. No patients with an UGA were included in 2014 in a clinical trial, versus 2 (2%) in 2015-2016. 13 patients with an UGA (12%) were treated in 2015-2016 with a MTT whereas in 2014, no patient (p = 0.001). CONCLUSIONS: In this retrospective analysis, we showed that the association of large-scale genomic testing and MTB was feasible, and could increase the prescription of MTT. However, in routine clinical practice, the majority of patients with UGA still do not have access to MTT.
Subject(s)
Molecular Targeted Therapy , Neoplasms/genetics , Neoplasms/therapy , Adolescent , Adult , Aged , Aged, 80 and over , Child , Drug Resistance, Neoplasm/genetics , Female , Health Services Accessibility , High-Throughput Nucleotide Sequencing/methods , Humans , Male , Medical Oncology , Middle Aged , Neoplasms/drug therapy , Precision Medicine/methods , Retrospective Studies , Treatment Outcome , Young AdultABSTRACT
Runt-related transcription factor 1 (RUNX1) is a well-known master regulator of hematopoietic lineages but its mechanisms of action are still not fully understood. Here, we found that RUNX1 localizes on active chromatin together with Far Upstream Binding Protein 1 (FUBP1) in human B-cell precursor lymphoblasts, and that both factors interact in the same transcriptional regulatory complex. RUNX1 and FUBP1 chromatin localization identified c-KIT as a common target gene. We characterized two regulatory regions, at +700 bp and +30 kb within the first intron of c-KIT, bound by both RUNX1 and FUBP1, and that present active histone marks. Based on these regions, we proposed a novel FUBP1 FUSE-like DNA-binding sequence on the +30 kb enhancer. We demonstrated that FUBP1 and RUNX1 cooperate for the regulation of the expression of the oncogene c-KIT. Notably, upregulation of c-KIT expression by FUBP1 and RUNX1 promotes cell proliferation and renders cells more resistant to the c-KIT inhibitor imatinib mesylate, a common therapeutic drug. These results reveal a new mechanism of action of RUNX1 that implicates FUBP1, as a facilitator, to trigger transcriptional regulation of c-KIT and to regulate cell proliferation. Deregulation of this regulatory mechanism may explain some oncogenic function of RUNX1 and FUBP1.
Subject(s)
Core Binding Factor Alpha 2 Subunit/genetics , DNA-Binding Proteins/genetics , Enhancer Elements, Genetic , Gene Expression Regulation, Leukemic , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Proto-Oncogene Proteins c-kit/genetics , RNA-Binding Proteins/genetics , Animals , Antineoplastic Agents/pharmacology , Base Sequence , Binding Sites , Bone Marrow Cells/drug effects , Bone Marrow Cells/metabolism , Bone Marrow Cells/pathology , Cell Line, Tumor , Cell Proliferation/drug effects , Chromatin/chemistry , Chromatin/metabolism , Core Binding Factor Alpha 2 Subunit/metabolism , DNA-Binding Proteins/metabolism , HEK293 Cells , Humans , Imatinib Mesylate/pharmacology , Mice , Mice, Inbred NOD , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Precursor Cells, B-Lymphoid/drug effects , Precursor Cells, B-Lymphoid/metabolism , Precursor Cells, B-Lymphoid/pathology , Primary Cell Culture , Protein Binding , Proto-Oncogene Proteins c-kit/metabolism , RNA-Binding Proteins/metabolism , Signal Transduction , Transcription, Genetic , Xenograft Model Antitumor AssaysABSTRACT
BRAF and MEK inhibitors (BRAFi and MEKi) are the standard of care for the treatment of metastatic melanoma in patients with BRAFV600E mutations, greatly improving progression-free survival. However, the acquisition of resistance to BRAFi and MEKi remains a difficult clinical challenge, with limited therapeutic options available for these patients. Here, we investigated the therapeutic potential of natural flavonoids as specific AhR (Aryl hydrocarbon Receptor) transcription factor antagonists in combination with BRAFi. EXPERIMENTAL DESIGN: Experiments were performed in vitro and in vivo with various human melanoma cell lines (mutated for BRAFV600E) sensitive or resistant to BRAFi. We evaluated the role of various flavonoids on cell sensitivity to BRAFi and their ability to counteract resistance and the invasive phenotype of melanoma. RESULTS: Flavonoids were highly effective in potentiating BRAFi therapy in human melanoma cell lines by increasing sensitivity and delaying the pool of resistant cells that arise during treatment. As AhR antagonists, flavonoids counteracted a gene expression program associated with the acquisition of resistance and phenotype switching that leads to an invasive and EMT-like phenotype. CONCLUSIONS: The use of natural flavonoids opens new therapeutic opportunities for the treatment of patients with BRAF-resistant disease.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/antagonists & inhibitors , Drug Resistance, Neoplasm/drug effects , Flavonoids/pharmacology , Melanoma/drug therapy , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Receptors, Aryl Hydrocarbon/antagonists & inhibitors , Animals , Basic Helix-Loop-Helix Transcription Factors/metabolism , Cell Line, Tumor , Chick Embryo , Humans , Melanoma/metabolism , Models, Molecular , Proto-Oncogene Proteins B-raf/metabolism , Receptors, Aryl Hydrocarbon/metabolismABSTRACT
Regarding several cytotoxic agents, it was evidenced that genetic polymorphisms in genes encoding enzymes involved in their metabolism are associated with higher risk of toxicity. Genotyping these genes before treatment is a valuable strategy to prevent side effects and to predict individual response to drug therapy. This pharmacogenetic approach is recommended for chemotherapies such as thiopurines (azathioprine, 6-mercaptopurine, thioguanine), irinotecan, and fluoropyrimidines (capecitabine and 5-fluorouracil). In this study, we aimed at developing and validating a fast, cost-effective, and easily implementable multiplex genotyping method suitable for analyzing a panel of nine variants involved in the pharmacogenetics of widely prescribed anticancer drugs. We designed a multiplex-specific PCR assay where fragments were labeled by two different fluorescent dye markers (HEX/FAM) identifiable by fragment analysis. These two labels were used to discriminate bi-allelic variants, while the size of the fragment allowed the identification of a particular polymorphism location. Variants of interest were TPMT (rs1800462, rs1142345, rs1800460), NUDT15 (rs116855232), DPYD (rs55886062, rs3918290, rs67376798, rs75017182), and UGT1A1 (rs8175347). The assay was repeatable, and genotypes could be determined when DNA sample amounts ranged from 25 to 100 ng. Primers and dye remained stable in a ready-to-use mixture solution after five freeze-thaw cycles. Accuracy was evidenced by the consistency of 187 genotyping results obtained with our multiplex assay and a reference method. The developed method is fast and cost-effective in simultaneously identifying nine variants involved in the pharmacological response of anticancer drugs. This assay can be easily implemented in laboratories for widespread access to pharmacogenetics in clinical practice.
Subject(s)
Antineoplastic Agents/pharmacology , Biomarkers, Tumor/genetics , Gene Expression Regulation, Neoplastic/drug effects , Neoplasms/pathology , Pharmacogenetics , Polymorphism, Single Nucleotide , Genetic Testing , Humans , Neoplasms/drug therapy , Neoplasms/geneticsABSTRACT
Targeting RNAs appears as an important opportunity to modulate biological processes. Here, we overviewed critical parameters implied in RNAs competition to bind small RNAs. These competitions influence small RNA availability and thereby gene expression and cell fate. We focused on the ability of RNAs to sequester small RNA, mainly the microRNAs (miRNAs) and proposed experimental workflows to demonstrate the existence and activity of RNA-sponge. From this basic science, we detailed tailored oligonucleotides, developed to challenge the binding of small RNA. In vitro and in vivo, these tailored oligonucleotides efficiently restore small RNA activity by preventing their sequestration on RNA-sponges.
Subject(s)
MicroRNAs/genetics , MicroRNAs/isolation & purification , Oligoribonucleotides, Antisense/metabolism , Arabidopsis/genetics , Arabidopsis/metabolism , Binding, Competitive , Cell Line, Tumor , Genes, Reporter , Hepatocytes/cytology , Hepatocytes/metabolism , Humans , Levivirus/chemistry , MicroRNAs/metabolism , Oligoribonucleotides, Antisense/chemical synthesis , Viral Proteins/genetics , Viral Proteins/metabolismABSTRACT
CD9, a member of the tetraspanin family, has been implicated in hematopoietic and leukemic stem cell homing. We investigated the role of CD9 in the dissemination of B acute lymphoblastic leukemia (B-ALL) cells, by stably downregulating CD9 in REH and NALM6 cells. CD9 expression was associated with higher levels of REH cell adhesion to fibronectin and C-X-C motif chemokine receptor 4 (CXCR4)-mediated migration. Death occurred later in NOD/SCID mice receiving REH cells depleted of CD9 for transplantation than in mice receiving control cells. After C-X-C motif chemokine ligand 12 (CXCL12) stimulation, CD9 promoted the formation of long cytoplasmic actin-rich protrusions. We demonstrated that CD9 enhanced RAC1 activation, in both REH cells and blasts from patients. Conversely, the overexpression of a competing CD9 C-terminal tail peptide in REH cytoplasm decreased RAC1 activation and cytoplasmic extension formation in response to CXCL12. Finally, the inhibition of RAC1 activation decreased migration in vitro, and the depletion of RAC1 protein from transplanted REH cells increased mouse survival. Furthermore, a testis-conditioned medium induced the migration of REH and NALM6 cells, and this migration was impeded by an anti-CD9 antibody. The level of CD9 expression also influenced the homing of these cells in mouse testes. These findings demonstrate, for the first time, that CD9 plays a key role in the CXCR4-mediated migration and engraftment of B-ALL cells in the bone marrow or testis, through RAC1 activation.
Subject(s)
Cell Movement , Gene Expression Regulation, Neoplastic , Neuropeptides/metabolism , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Receptors, CXCR4/metabolism , Tetraspanin 29/metabolism , rac1 GTP-Binding Protein/metabolism , Animals , Apoptosis , Blotting, Western , Bone Marrow/metabolism , Bone Marrow/pathology , Cell Adhesion , Cell Proliferation , Chemokine CXCL12/metabolism , Humans , Immunoprecipitation , Male , Mice , Mice, Inbred NOD , Mice, SCID , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , Testis/metabolism , Testis/pathology , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
Genomic instability is a major hallmark of cancer. To maintain genomic integrity, cells are equipped with dedicated sensors to monitor DNA repair or to force damaged cells into death programs. The tumor suppressor p53 is central in this process. Here, we report that the ubiquitous transcription factor Upstream Stimulatory factor 1 (USF1) coordinates p53 function in making proper cell fate decisions. USF1 stabilizes the p53 protein and promotes a transient cell cycle arrest, in the presence of DNA damage. Thus, cell proliferation is maintained inappropriately in Usf1 KO mice and in USF1-deficient melanoma cells challenged by genotoxic stress. We further demonstrate that the loss of USF1 compromises p53 stability by enhancing p53-MDM2 complex formation and MDM2-mediated degradation of p53. In USF1-deficient cells, the level of p53 can be restored by the re-expression of full-length USF1 protein similarly to what is observed using Nutlin-3, a specific inhibitor that prevents p53-MDM2 interaction. Consistent with a new function for USF1, a USF1 truncated protein lacking its DNA-binding and transactivation domains can also restore the induction and activity of p53. These findings establish that p53 function requires the ubiquitous stress sensor USF1 for appropriate cell fate decisions in response to DNA-damage. They underscore the new role of USF1 and give new clues of how p53 loss of function can occur in any cell type. Finally, these findings are of clinical relevance because they provide new therapeutic prospects in stabilizing and reactivating the p53 pathway.
Subject(s)
Cell Differentiation , Neoplasms/genetics , Tumor Suppressor Protein p53/metabolism , Upstream Stimulatory Factors/metabolism , Animals , Apoptosis/genetics , Cell Line, Tumor , Cell Lineage , Cell Proliferation , DNA Damage/genetics , Genomic Instability , Humans , Mice , Protein Interaction Maps/genetics , Proto-Oncogene Proteins c-mdm2/metabolism , Tumor Suppressor Protein p53/genetics , Upstream Stimulatory Factors/geneticsABSTRACT
The stress-activated p38α MAP Kinase is an integral and critical component of the UV-induced inflammatory response. Despite the advances in recent years in the development of p38 kinase inhibitors, validation of these compounds in the diseased models remains limited. Based on the pharmacological profile of p38α inhibitor lead compound, SB203580, we synthesized a series of pyrrole-derivatives. Using UV-irradiated human skin punch-biopsies and cell cultures, we identified and validated the inhibitory activity of the derivatives by quantitatively measuring their effect on the expression of p38α target genes using real-time PCR. This approach not only identified pyrrole-2 as a unique derivative of this series that specifically inhibited the UV-activated p38α kinase, but also documented the skin permeation, bioavailability and reversible properties of this derivative in a 3D structure. The successful skin permeation of pyrrole-2 and its impact on AREG, COX-2 and MMP-9 gene expression demonstrates its potential use in modulating inflammatory processes in the skin. This study underscored the importance of using adapted biological models to identify accurate bioactive compounds.
Subject(s)
Gene Expression/drug effects , Protein Kinase Inhibitors/pharmacology , Skin/enzymology , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , Amphiregulin/genetics , Cells, Cultured , Cyclooxygenase 2/genetics , Humans , Matrix Metalloproteinase 9/genetics , Models, Biological , Pyrroles/chemistry , Pyrroles/pharmacology , Real-Time Polymerase Chain Reaction , Ultraviolet Rays , p38 Mitogen-Activated Protein Kinases/genetics , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
An important function of all organisms is to ensure that their genetic material remains intact and unaltered through generations. This is an extremely challenging task since the cell's DNA is constantly under assault by endogenous and environmental agents. To protect against this, cells have evolved effective mechanisms to recognize DNA damage, signal its presence, and mediate its repair. While these responses are expected to be highly regulated because they are critical to avoid human diseases, very little is known about the regulation of the expression of genes involved in mediating their effects. The Nucleotide Excision Repair (NER) is the major DNA-repair process involved in the recognition and removal of UV-mediated DNA damage. Here we use a combination of in vitro and in vivo assays with an intermittent UV-irradiation protocol to investigate the regulation of key players in the DNA-damage recognition step of NER sub-pathways (TCR and GGR). We show an up-regulation in gene expression of CSA and HR23A, which are involved in TCR and GGR, respectively. Importantly, we show that this occurs through a p53 independent mechanism and that it is coordinated by the stress-responsive transcription factor USF-1. Furthermore, using a mouse model we show that the loss of USF-1 compromises DNA repair, which suggests that USF-1 plays an important role in maintaining genomic stability.
Subject(s)
DNA Damage/genetics , DNA Repair/genetics , DNA/genetics , Upstream Stimulatory Factors/genetics , Animals , Biopsy , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Survival/radiation effects , DNA/radiation effects , DNA Repair Enzymes/genetics , DNA Repair Enzymes/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Gene Expression Regulation/radiation effects , Genomic Instability , HSP70 Heat-Shock Proteins/genetics , HSP70 Heat-Shock Proteins/metabolism , Humans , Keratinocytes/cytology , Keratinocytes/metabolism , Keratinocytes/radiation effects , Mice , Mice, Knockout , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , RNA, Small Interfering , Ultraviolet RaysABSTRACT
Merkel cell carcinoma is believed to be derived from Merkel cells after infection by Merkel cell polyomavirus (MCPyV) and other poorly understood events. Transcriptional profiling using cDNA microarrays was performed on cells from MCPy-negative and MCPy-positive Merkel cell carcinomas and isolated normal Merkel cells. This microarray revealed numerous significantly upregulated genes and some downregulated genes. The extensive list of genes that were identified in these experiments provides a large body of potentially valuable information of Merkel cell carcinoma carcinogenesis and could represent a source of potential targets for cancer therapy.
Subject(s)
Carcinoma, Merkel Cell/genetics , Merkel Cells/metabolism , Skin Neoplasms/genetics , Carcinoma, Merkel Cell/virology , Gene Expression Profiling , Gene Regulatory Networks , Humans , Merkel cell polyomavirus/isolation & purification , Oligonucleotide Array Sequence Analysis , Polyomavirus Infections/genetics , Polyomavirus Infections/virology , Skin Neoplasms/virology , Tumor Virus Infections/genetics , Tumor Virus Infections/virologyABSTRACT
The COVID-19 crisis and the development of the first approved mRNA vaccine have highlighted the power of RNA-based therapeutic strategies for the development of new medicines. Aside from RNA-vaccines, antisense oligonucleotides (ASOs) represent a new and very promising class of RNA-targeted therapy. Few drugs have already received approval from the Food and Drug Administration. Here, we underscored why and how ASOs hold the potential to change the therapeutic landscape to beat SARS-CoV-2 viral infections. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Small Molecule-RNA Interactions.
Subject(s)
COVID-19 Drug Treatment , Oligonucleotides, Antisense , Humans , Oligonucleotides , Oligonucleotides, Antisense/genetics , Oligonucleotides, Antisense/pharmacology , RNA , SARS-CoV-2 , United States , Vaccines, Synthetic , mRNA VaccinesABSTRACT
The nongenetic mechanisms required to control tumor phenotypic plasticity and shape drug-resistance remain unclear. We show here that the Aryl hydrocarbon Receptor (AhR) transcription factor directly regulates the gene expression program associated with the acquisition of resistance to BRAF inhibitor (BRAFi) in melanoma. In addition, we show in melanoma cells that canonical activation of AhR mediates the activation of the SRC pathway and promotes the acquisition of an invasive and aggressive resistant phenotype to front-line BRAFi treatment in melanoma. This nongenetic reprogramming identifies a clinically compatible approach to reverse BRAFi resistance in melanoma. Using a preclinical BRAFi-resistant PDX melanoma model, we demonstrate that SRC inhibition with dasatinib significantly re-sensitizes melanoma cells to BRAFi. Together we identify the AhR/SRC axis as a new therapeutic vulnerability to trigger resistance and warrant the introduction of SRC inhibitors during the course of the treatment in combination with front-line therapeutics to delay BRAFi resistance.
Subject(s)
Melanoma , Receptors, Aryl Hydrocarbon , Humans , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Dasatinib/pharmacology , Dasatinib/therapeutic use , Melanoma/drug therapy , PhenotypeABSTRACT
In the field of pharmacogenetics, the trend is to analyze a panel of several actionable genetic polymorphisms. It may require the use of high-throughput sequencing which demands expensive reagents/instruments and specific skills to interpret results. As an alternative, the aim of this work was to validate an easy, fast, and inexpensive multiplex pharmacogenetics assay to simultaneously genotype a panel of 17 clinically actionable variants involved in drug pharmacokinetics/pharmacodynamics. We designed primers to perform a multiplex PCR assay using a single mix. Primers were labeled by two fluorescent dye markers to discriminate alleles, while the size of the PCR fragments analyzed by electrophoresis allowed identifying amplicon. Polymorphisms of interest were CYP3A4*22, CYP3A5*3, CYP1A2*1F, CYP2C9*2-*3, CYP2C19*2-*3-*17, VKORC1-1639G > A, ABCB1 rs1045642-rs1128503-rs2229109-rs2032582, and CYP2D6*3-*4-*6-*9. The assay was repeatable and a minimum quantity of 10 ng of DNA/ sample was needed to obtain accurate results. The method was applied to a validation cohort of 121 samples and genotyping results were consistent with those obtained with reference methods. The assay was fast and cost-effective with results being available within one working-day. This robust assay can easily be implemented in laboratories as an alternative to cumbersome simplex assays or expensive multiplex approaches. Together it should widespread access to pharmacogenetics in clinical routine practice.
ABSTRACT
Acute lymphoblastic leukemias (ALL) are the most frequent cancer in children and derive most often from B-cell precursors. Current survival rates roughly reach 90% at 10 years from diagnosis. However, 15-20% of children still relapse with a significant risk of death. Our previous work showed that the transmembrane protein CD9 plays a major role in lymphoblasts migration into sanctuary sites, especially in testis, through the activation of RAC1 signaling upon blasts stimulation with C-X-C chemokine ligand 12 (CXCL12). Here, we identified common factors shared by the bone marrow and extramedullary niches which could upregulate CD9 expression and function. We found that low oxygen levels enhance CD9 expression both at mRNA and protein levels. We further determined that Hypoxia Inducible Factor 1α (HIF1α), the master transcription factor involved in hypoxia response, binds directly CD9 promoter and induce CD9 transcription. We also showed that CD9 protein is crucial for leukemic cell adhesion and migration at low oxygen levels, possibly through its action on RAC1 signaling. Mouse xenograft experiments indicate that HIF1α signaling pathway promotes ALL cells engraftment in a CD9-dependent manner. The present work increments our understanding of CD9 implication in ALL pathogenesis.
Subject(s)
Hypoxia , Signal Transduction , Male , Humans , Mice , Animals , Tetraspanin 29/genetics , Tetraspanin 29/metabolism , Cell Adhesion , OxygenABSTRACT
Uveal melanoma (UM), the most common primary intraocular tumor in adults, has been extensively characterized by omics technologies during the last 5 yr. Despite the discovery of gene signatures, the molecular actors driving cancer aggressiveness are not fully understood, and UM is still associated with very poor overall survival (OS) at the metastatic stage. By defining the miR-16 interactome, we revealed that miR-16 mainly interacts via non-canonical base-pairing to a subset of RNAs, promoting their expression levels. Consequently, the canonical miR-16 activity, involved in the RNA decay of oncogenes, such as <i>cyclin D3</i>, is impaired. This non-canonical base-pairing can explain both the derepression of miR-16 targets and the promotion of oncogene expression observed in patients with poor OS in two cohorts. miR-16 activity, assessment using our RNA signature, discriminates the patient's OS as effectively as current methods. To the best of our knowledge, this is the first time that a predictive signature has been composed of genes belonging to the same mechanism (miR-16) in UM. Altogether, our results strongly suggest that UM is a miR-16 disease.
Subject(s)
Melanoma , MicroRNAs , Uveal Neoplasms , Adult , Base Pairing , Cyclin D3 , Humans , Melanoma/metabolism , MicroRNAs/genetics , MicroRNAs/metabolism , Uveal Neoplasms/genetics , Uveal Neoplasms/metabolism , Uveal Neoplasms/pathologyABSTRACT
Because of a narrow therapeutic index and a wide inter- and intra-patient variability, therapeutic drug monitoring of the immunosuppressant drug tacrolimus (TAC) based on whole-blood concentrations (Cblood) is mandatory in solid organ transplant recipients. Using peripheral blood mononuclear cells concentrations (CPBMC) could improve patient outcomes. The poor correlation between Cblood and CPBMC makes hypothesize that drug transporters are implicated in the intracellular accumulation of TAC. The aim of this work was therefore to clinically study: i) the role of genetic variants and ii) the effect of mRNA and protein expression of 4 drug transporters on the TAC CPBMC/blood ratio. In addition, functional in vitro experiments were performed to mechanistically validate the clinical observations. Genetic variants of ABCB1/P-gp and SLC28A3/CNT3 did not influence TAC CPBMC in liver transplant recipients (LTR). ABCC2/MRP2 at the mRNA level; ABCB1/P-gp, SLC28A3/CNT3 and SLC29A1/ENT1 at the protein level; correlated with the CPBMC/blood in kidney and LTR. In vitro results suing transporter-expressing cells confirmed that TAC is substrate of P-gp but not MRP2, whereas experiments remained inconclusive for CNT3 and ENT1. In conclusion, the genetic-transcription-protein-functional approach presented in this work provides new insights in the understanding of TAC transport at the T lymphocyte plasma membrane.
Subject(s)
Liver Transplantation , Tacrolimus , Humans , Leukocytes, Mononuclear/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Cytochrome P-450 CYP3A/metabolism , T-Lymphocytes , Immunosuppressive Agents , KidneyABSTRACT
BACKGROUND: Low miR-31-3p expression was identified as predictive of anti-EGFR efficacy in RAS-wt mCRC. Primary tumor side was also proposed as a predictive factor of anti-EGFR benefit. This retrospective multicentric study evaluated the predictive role of miR-31-3p in right-sided RAS-wt mCRC patients treated with first-line CT+anti-EGFR or CT+bevacizumab (Beva). METHODS: Seventy-two right-sided RAS-wt mCRC patients treated in first-line with CT+anti-EGFR (n = 43) or Beva (n = 29) were included. Overall survival (OS), progression-free survival (PFS) and response rate (RR) were analyzed and stratified according to tumor miR-31-3p expression level and targeted therapy (TT). RESULTS: BRAF V600E mutation was more frequent in high vs low miR-31-3p expressers (60.6% vs 15.4%, P < 0.001). PFS was significantly longer with CT+Beva than with CT+anti-EGFR (13 vs 7 months; P = 0.024). Among low miR-31-3p expressers, PFS, OS and RR were not significantly different between the two groups, while in high miR-31-3p expressers, only PFS was longer in the CT+Beva group (11 vs 6 months; P = 0.03). In patients treated with CT+anti-EGFR, low miR-31-3p expressers had a significantly longer OS (20 vs 13 months; P = 0.02) than high miR-31-3p expressers. ORR was not significantly different between the two groups of treatment, in both low and high miR-31-3p expressers. MiR-31-3p expression status was statistically correlated between primary tumors and corresponding metastases. CONCLUSION: In this study, miR-31-3p couldn't identify a subgroup of patients with right-sided RAS-wt mCRC who might benefit from anti-EGFR and suggest that Beva is the TT of choice in first-line treatment of these patients.