ABSTRACT
Deregulated metabolism is one of the hallmarks of cancer. It is well-known that tumour cells tend to metabolize glucose via glycolysis even when oxygen is available and mitochondrial respiration is functional. However, the lower energy efficiency of aerobic glycolysis with respect to mitochondrial respiration makes this behaviour, namely the Warburg effect, counter-intuitive, although it has now been recognized as source of anabolic precursors. On the other hand, there is evidence that oxygenated tumour cells could be fuelled by exogenous lactate produced from glycolysis. We employed a multi-scale approach that integrates multi-agent modelling, diffusion-reaction, stoichiometric equations, and Boolean networks to study metabolic cooperation between hypoxic and oxygenated cells exposed to varying oxygen, nutrient, and inhibitor concentrations. The results show that the cooperation reduces the depletion of environmental glucose, resulting in an overall advantage of using aerobic glycolysis. In addition, the oxygen level was found to be decreased by symbiosis, promoting a further shift towards anaerobic glycolysis. However, the oxygenated and hypoxic populations may gradually reach quasi-equilibrium. A sensitivity analysis using Latin hypercube sampling and partial rank correlation shows that the symbiotic dynamics depends on properties of the specific cell such as the minimum glucose level needed for glycolysis. Our results suggest that strategies that block glucose transporters may be more effective to reduce tumour growth than those blocking lactate intake transporters.
Subject(s)
Neoplasms , Symbiosis , Humans , Glycolysis , Lactic Acid/metabolism , Neoplasms/metabolism , Glucose/metabolism , Hypoxia , OxygenABSTRACT
Chromosomal instability (CIN) drives cell-to-cell heterogeneity, and the development of genetic diseases, including cancer. Impaired homologous recombination (HR) has been implicated as a major driver of CIN, however, the underlying mechanism remains unclear. Using a fission yeast model system, we establish a common role for HR genes in suppressing DNA double-strand break (DSB)-induced CIN. Further, we show that an unrepaired single-ended DSB arising from failed HR repair or telomere loss is a potent driver of widespread CIN. Inherited chromosomes carrying a single-ended DSB are subject to cycles of DNA replication and extensive end-processing across successive cell divisions. These cycles are enabled by Cullin 3-mediated Chk1 loss and checkpoint adaptation. Subsequent propagation of unstable chromosomes carrying a single-ended DSB continues until transgenerational end-resection leads to fold-back inversion of single-stranded centromeric repeats and to stable chromosomal rearrangements, typically isochromosomes, or to chromosomal loss. These findings reveal a mechanism by which HR genes suppress CIN and how DNA breaks that persist through mitotic divisions propagate cell-to-cell heterogeneity in the resultant progeny.
Subject(s)
Schizosaccharomyces , Humans , Chromosomal Instability , DNA Breaks, Double-Stranded , DNA Repair , Homologous Recombination , Schizosaccharomyces/genetics , Schizosaccharomyces/metabolismABSTRACT
The intratumor microenvironment generates phenotypically distinct but interconvertible malignant cell subpopulations that fuel metastatic spread and therapeutic resistance. Whether different microenvironmental cues impose invasive or therapy-resistant phenotypes via a common mechanism is unknown. In melanoma, low expression of the lineage survival oncogene microphthalmia-associated transcription factor (MITF) correlates with invasion, senescence, and drug resistance. However, how MITF is suppressed in vivo and how MITF-low cells in tumors escape senescence are poorly understood. Here we show that microenvironmental cues, including inflammation-mediated resistance to adoptive T-cell immunotherapy, transcriptionally repress MITF via ATF4 in response to inhibition of translation initiation factor eIF2B. ATF4, a key transcription mediator of the integrated stress response, also activates AXL and suppresses senescence to impose the MITF-low/AXL-high drug-resistant phenotype observed in human tumors. However, unexpectedly, without translation reprogramming an ATF4-high/MITF-low state is insufficient to drive invasion. Importantly, translation reprogramming dramatically enhances tumorigenesis and is linked to a previously unexplained gene expression program associated with anti-PD-1 immunotherapy resistance. Since we show that inhibition of eIF2B also drives neural crest migration and yeast invasiveness, our results suggest that translation reprogramming, an evolutionarily conserved starvation response, has been hijacked by microenvironmental stress signals in melanoma to drive phenotypic plasticity and invasion and determine therapeutic outcome.
Subject(s)
Cell Plasticity/genetics , Cellular Reprogramming/genetics , Drug Resistance, Neoplasm/genetics , Gene Expression Regulation, Neoplastic/genetics , Melanoma/genetics , Microphthalmia-Associated Transcription Factor/genetics , Protein Biosynthesis/genetics , Animals , Cellular Microenvironment , Evolution, Molecular , Feedback, Physiological , Gene Expression Regulation, Neoplastic/drug effects , Glutamine/pharmacology , Humans , Immunotherapy , Melanoma/drug therapy , Melanoma/metabolism , Neoplasm Invasiveness/genetics , Neural Crest/cytology , Phenotype , Transcription Factors/metabolism , Zebrafish/embryologyABSTRACT
BACKGROUND: It is important to identify molecular features that improve prostate cancer (PCa) risk stratification before radical treatment with curative intent. Molecular analysis of historical diagnostic formalin-fixed paraffin-embedded (FFPE) prostate biopsies from cohorts with post-radiotherapy (RT) long-term clinical follow-up has been limited. Utilizing parallel sequencing modalities, we performed a proof-of-principle sequencing analysis of historical diagnostic FFPE prostate biopsies. We compared patients with (i) stable PCa (sPCa) postprimary or salvage RT, (ii) progressing PCa (pPCa) post-RT, and (iii) de novo metastatic PCa (mPCa). METHODS: A cohort of 19 patients with diagnostic prostate biopsies (n = 6 sPCa, n = 5 pPCa, n = 8 mPCa) and mean 4 years 10 months follow-up (diagnosed 2009-2016) underwent nucleic acid extraction from demarcated malignancy. Samples underwent 3'RNA sequencing (3'RNAseq) (n = 19), nanoString analysis (n = 12), and Illumina 850k methylation (n = 8) sequencing. Bioinformatic analysis was performed to coherently identify differentially expressed genes and methylated genomic regions (MGRs). RESULTS: Eighteen of 19 samples provided useable 3'RNAseq data. Principal component analysis (PCA) demonstrated similar expression profiles between pPCa and mPCa cases, versus sPCa. Coherently differentially methylated probes between these groups identified ~600 differentially MGRs. The top 50 genes with increased expression in pPCa patients were associated with reduced progression-free survival post-RT (p < 0.0001) in an external cohort. CONCLUSIONS: 3'RNAseq, nanoString and 850k-methylation analyses are each achievable from historical FFPE diagnostic pretreatment prostate biopsies, unlocking the potential to utilize large cohorts of historic clinical samples. Profiling similarities between individuals with pPCa and mPCa suggests biological similarities and historical radiological staging limitations, which warrant further investigation.
Subject(s)
Disease Progression , Prostatic Neoplasms , Humans , Male , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Prostatic Neoplasms/radiotherapy , Aged , Middle Aged , Biopsy , Genomics , Prostate/pathology , Neoplasm Metastasis , Cohort StudiesABSTRACT
The effect of radiation therapy on tumor vasculature has long been a subject of debate. Increased oxygenation and perfusion have been documented during radiation therapy. Conversely, apoptosis of endothelial cells in irradiated tumors has been proposed as a major contributor to tumor control. To examine these contradictions, we use multiphoton microscopy in two murine tumor models: MC38, a highly vascularized, and B16F10, a moderately vascularized model, grown in transgenic mice with tdTomato-labeled endothelium before and after a single (15 Gy) or fractionated (5 × 3 Gy) dose of radiation. Unexpectedly, even these high doses lead to little structural change of the perfused vasculature. Conversely, non-perfused vessels and blind ends are substantially impaired after radiation accompanied by apoptosis and reduced proliferation of their endothelium. RNAseq analysis of tumor endothelial cells confirms the modification of gene expression in apoptotic and cell cycle regulation pathways after irradiation. Therefore, we conclude that apoptosis of tumor endothelial cells after radiation does not impair vascular structure.
Subject(s)
Endothelial Cells , Neoplasms , Animals , Apoptosis , Endothelial Cells/metabolism , Endothelium/metabolism , Mice , Mice, Transgenic , Neoplasms/genetics , Neoplasms/metabolism , Neoplasms/radiotherapy , Radiation, IonizingABSTRACT
Almost all biomedical research to date has relied upon mean measurements from cell populations, however it is well established that what it is observed at this macroscopic level can be the result of many interactions of several different single cells. Thus, the observable macroscopic 'average' cannot outright be used as representative of the 'average cell'. Rather, it is the resulting emerging behaviour of the actions and interactions of many different cells. Single-cell RNA sequencing (scRNA-Seq) enables the comparison of the transcriptomes of individual cells. This provides high-resolution maps of the dynamic cellular programmes allowing us to answer fundamental biological questions on their function and evolution. It also allows to address medical questions such as the role of rare cell populations contributing to disease progression and therapeutic resistance. Furthermore, it provides an understanding of context-specific dependencies, namely the behaviour and function that a cell has in a specific context, which can be crucial to understand some complex diseases, such as diabetes, cardiovascular disease and cancer. Here, we provide an overview of scRNA-Seq, including a comparative review of emerging technologies and computational pipelines. We discuss the current and emerging applications and focus on tumour heterogeneity a clear example of how scRNA-Seq can provide new understanding of a complex disease. Additionally, we review the limitations and highlight the need of powerful computational pipelines and reproducible protocols for the broader acceptance of this technique in basic and clinical research.
Subject(s)
Neoplasms , Single-Cell Analysis , Humans , Single-Cell Analysis/methods , Transcriptome/genetics , Neoplasms/genetics , RNA/genetics , TechnologyABSTRACT
Obesity-related cancers account for 40% of the cancer cases observed in the USA and obesity is overtaking smoking as the most widespread modifiable risk factor for carcinogenesis. Here, we use the hallmarks of cancer framework to delineate how obesity might influence the carcinogenic hallmarks in somatic cells. We discuss the effects of obesity on (a) sustaining proliferative signaling; (b) evading growth suppressors; (c) resisting cell death; (d) enabling replicative immortality; (e) inducing angiogenesis; (f) activating invasion and metastasis; (g) reprogramming energy metabolism; and (h) avoiding immune destruction, together with its effects on genome instability and tumour-promoting inflammation. We present the current understanding and controversies in this evolving field, and highlight some areas in need of further cross-disciplinary focus. For instance, the relative importance of the many potentially causative obesity-related factors is unclear for each type of malignancy. Even within a single tumour type, it is currently unknown whether one obesity-related factor consistently plays a predominant role, or if this varies between patients or, even in a single patient with time. Clarifying how the hallmarks are affected by obesity may lead to novel prevention and treatment strategies for the increasingly obese population.
Subject(s)
Carcinogenesis , Neoplasms , Humans , Neoplasms/metabolism , Neovascularization, Pathologic/pathology , Obesity/complications , Signal TransductionABSTRACT
Cyclins are central engines of cell cycle progression in conjunction with cyclin-dependent kinases (CDKs). Among the different cyclins controlling cell cycle progression, cyclin F does not partner with a CDK, but instead forms via its F-box domain an SCF (Skp1-Cul1-F-box)-type E3 ubiquitin ligase module. Although various substrates of cyclin F have been identified, the vulnerabilities of cells lacking cyclin F are not known. Thus, we assessed viability of cells lacking cyclin F upon challenging them with more than 180 different kinase inhibitors. The screen revealed a striking synthetic lethality between Chk1 inhibition and cyclin F loss. Chk1 inhibition in cells lacking cyclin F leads to DNA replication catastrophe. Replication catastrophe depends on accumulation of the transcription factor E2F1 in cyclin F-depleted cells. We find that SCF-cyclin F controls E2F1 ubiquitylation and degradation during the G2/M phase of the cell cycle and upon challenging cells with Chk1 inhibitors. Thus, Cyclin F restricts E2F1 activity during the cell cycle and upon checkpoint inhibition to prevent DNA replication stress. Our findings pave the way for patient selection in the clinical use of checkpoint inhibitors.
Subject(s)
Checkpoint Kinase 1/antagonists & inhibitors , Cyclins/metabolism , E2F1 Transcription Factor/metabolism , Protein Kinase Inhibitors/pharmacology , Proteolysis , SKP Cullin F-Box Protein Ligases/metabolism , Synthetic Lethal Mutations , Cell Cycle/drug effects , Checkpoint Kinase 1/genetics , Cyclins/genetics , DNA Replication , E2F1 Transcription Factor/genetics , HeLa Cells , Humans , Phosphorylation , Protein Binding , SKP Cullin F-Box Protein Ligases/genetics , UbiquitinationABSTRACT
SETD2-dependent H3 Lysine-36 trimethylation (H3K36me3) has been recently linked to the deposition of de-novo DNA methylation. SETD2 is frequently mutated in cancer, however, the functional impact of SETD2 loss and depletion on DNA methylation across cancer types and tumorigenesis is currently unknown. Here, we perform a pan-cancer analysis and show that both SETD2 mutation and reduced expression are associated with DNA methylation dysregulation across 21 out of the 24 cancer types tested. In renal cancer, these DNA methylation changes are associated with altered gene expression of oncogenes, tumour suppressors, and genes involved in neoplasm invasiveness, including TP53, FOXO1, and CDK4. This suggests a new role for SETD2 loss in tumorigenesis and cancer aggressiveness through DNA methylation dysregulation. Moreover, using a robust machine learning methodology, we develop and validate a 3-CpG methylation signature which is sufficient to predict SETD2 mutation status with high accuracy and correlates with patient prognosis.
Subject(s)
Carcinoma, Renal Cell , Kidney Neoplasms , Humans , DNA Methylation , Histones/metabolism , Carcinoma, Renal Cell/pathology , Kidney Neoplasms/pathology , Carcinogenesis/genetics , Cell Transformation, Neoplastic/geneticsABSTRACT
ChipCytometry is a multiplex imaging method that can be used to analyze either cell suspensions or tissue sections. Images are acquired by iterative cycles of immunostaining with fluorescently labeled Abs, followed by photobleaching, which allows the accumulation of multiple markers on a single sample. In this study, we explored the feasibility of using ChipCytometry to identify and phenotype cell subsets, including rare cell types, using a combination of tissue sections and single-cell suspensions. Using ChipCytometry of tissue sections, we successfully demonstrated the architecture of human palatine tonsils, including the B and T cell zones, and characterized subcompartments such as the B cell mantle and germinal center zone, as well as intrafollicular PD1-expressing CD4+ T cells. Additionally, we were able to identify the rare tonsillar T cell subsets, mucosal-associated invariant T (MAIT) and γδ-T cells, within tonsil tissue. Using single-cell suspension ChipCytometry, we further dissected human tonsillar T cell subsets via unsupervised clustering analysis as well as supervised traditional manual gating. We were able to show that PD1+CD4+ T cells are comprised of CXCR5+BCL6high follicular Th cells and CXCR5-BCL6mid pre-follicular Th cells. Both supervised and unsupervised analysis approaches identified MAIT cells in single-cell suspensions, confirming a phenotype similar to that of blood-derived MAIT cells. In this study, we demonstrate that ChipCytometry is a viable method for single-cell suspension cytometry and analysis, with the additional benefit of allowing phenotyping in a spatial context using tissue sections.
Subject(s)
Germinal Center , Palatine Tonsil , B-Lymphocytes , Humans , T-Lymphocyte Subsets , T-Lymphocytes, Helper-InducerABSTRACT
Single-cell RNA sequencing experiments produce data useful to identify different cell types, including uncharacterized and rare ones. This enables us to study the specific functional roles of these cells in different microenvironments and contexts. After identifying a (novel) cell type of interest, it is essential to build succinct marker panels, composed of a few genes referring to cell surface proteins and clusters of differentiation molecules, able to discriminate the desired cells from the other cell populations. In this work, we propose a fully-automatic framework called MAGNETO, which can help construct optimal marker panels starting from a single-cell gene expression matrix and a cell type identity for each cell. MAGNETO builds effective marker panels solving a tailored bi-objective optimization problem, where the first objective regards the identification of the genes able to isolate a specific cell type, while the second conflicting objective concerns the minimization of the total number of genes included in the panel. Our results on three public datasets show that MAGNETO can identify marker panels that identify the cell populations of interest better than state-of-the-art approaches. Finally, by fine-tuning MAGNETO, our results demonstrate that it is possible to obtain marker panels with different specificity levels.
Subject(s)
Single-Cell Analysis , Transcriptome , Sequence Analysis, RNA/methods , Single-Cell Analysis/methods , Gene Expression Profiling/methods , Cell DifferentiationABSTRACT
Tumor heterogeneity includes variable and fluctuating oxygen concentrations, which result in the accumulation of hypoxic regions in most solid tumors. Tumor hypoxia leads to increased therapy resistance and has been linked to genomic instability. Here, we tested the hypothesis that exposure to levels of hypoxia that cause replication stress could increase APOBEC activity and the accumulation of APOBEC-mediated mutations. APOBEC-dependent mutational signatures have been well-characterized, although the physiological conditions which underpin them have not been described. We demonstrate that fluctuating/cyclic hypoxic conditions which lead to replication catastrophe induce the expression and activity of APOBEC3B. In contrast, stable/chronic hypoxic conditions which induce replication stress in the absence of DNA damage are not sufficient to induce APOBEC3B. Most importantly, the number of APOBEC-mediated mutations in patient tumors correlated with a hypoxia signature. Together, our data support the conclusion that hypoxia-induced replication catastrophe drives genomic instability in tumors, specifically through increasing the activity of APOBEC3B.
Subject(s)
Cytidine Deaminase/metabolism , DNA Replication , Minor Histocompatibility Antigens/metabolism , Neoplasms/enzymology , APOBEC Deaminases/metabolism , Cell Hypoxia , Cell Line, Tumor , Deamination , Humans , Hydroxyurea/toxicity , Stress, Physiological/geneticsABSTRACT
PURPOSE OF REVIEW: Gliomas are the most common primary tumors of the central nervous system. They are characterized by a disappointing prognosis and ineffective therapy that has shown no substantial improvements in the past 20âyears. The lack of progress in treating gliomas is linked with the inadequacy of suitable tumor samples to plan translational studies and support laboratory developments. To overcome the use of tumor tissue, this commentary review aims to highlight the potential for the clinical application of liquid biopsy (intended as the study of circulating biomarkers in the blood), focusing on circulating tumor cells, circulating DNA and circulating noncoding RNA. RECENT FINDINGS: Thanks to the increasing sensitivity of sequencing techniques, it is now possible to analyze circulating nucleic acids and tumor cells (liquid biopsy). SUMMARY: Although studies on the use of liquid biopsy are still at an early stage, the potential clinical applications of liquid biopsy in the study of primary brain cancer are many and have the potential to revolutionize the approach to neuro-oncology, and importantly, they offer the possibility of gathering information on the disease at any time during its history.
Subject(s)
Cell-Free Nucleic Acids , Glioma , Neoplastic Cells, Circulating , Biomarkers, Tumor/genetics , Cell-Free Nucleic Acids/genetics , Glioma/diagnosis , Humans , Liquid Biopsy/methods , Neoplastic Cells, Circulating/pathology , RNA, UntranslatedABSTRACT
BACKGROUND: Height and other anthropometric measures are consistently found to associate with differential cancer risk. However, both genetic and mechanistic insights into these epidemiological associations are notably lacking. Conversely, inherited genetic variants in tumour suppressors and oncogenes increase cancer risk, but little is known about their influence on anthropometric traits. METHODS: By integrating inherited and somatic cancer genetic data from the Genome-Wide Association Study Catalog, expression Quantitative Trait Loci databases and the Cancer Gene Census, we identify SNPs that associate with different cancer types and differential gene expression in at least one tissue type, and explore the potential pleiotropic associations of these SNPs with anthropometric traits through SNP-wise association in a cohort of 500,000 individuals. RESULTS: We identify three regulatory SNPs for three important cancer genes, FANCA, MAP3K1 and TP53 that associate with both anthropometric traits and cancer risk. Of particular interest, we identify a previously unrecognised strong association between the rs78378222[C] SNP in the 3' untranslated region (3'-UTR) of TP53 and both increased risk for developing non-melanomatous skin cancer (OR=1.36 (95% 1.31 to 1.41), adjusted p=7.62E-63), brain malignancy (OR=3.12 (2.22 to 4.37), adjusted p=1.43E-12) and increased standing height (adjusted p=2.18E-24, beta=0.073±0.007), lean body mass (adjusted p=8.34E-37, beta=0.073±0.005) and basal metabolic rate (adjusted p=1.13E-31, beta=0.076±0.006), thus offering a novel genetic link between these anthropometric traits and cancer risk. CONCLUSION: Our results clearly demonstrate that heritable variants in key cancer genes can associate with both differential cancer risk and anthropometric traits in the general population, thereby lending support for a genetic basis for linking these human phenotypes.
Subject(s)
Body Weights and Measures , Neoplasms/genetics , Oncogenes , Polymorphism, Single Nucleotide , Adult , Aged , Anthropometry , Cohort Studies , Female , Genetic Linkage , Genetic Pleiotropy , Genetic Predisposition to Disease , Genome-Wide Association Study , Humans , Male , Middle Aged , Quantitative Trait Loci , Quantitative Trait, Heritable , Risk AssessmentABSTRACT
miRNA are regulators of cell phenotype, and there is clear evidence that these small posttranscriptional modifiers of gene expression are involved in defining a cellular response across states of development and disease. Classical methods for elucidating the repressive effect of a miRNA on its targets involve controlling for the many factors influencing miRNA action, and this can be achieved in cell lines, but misses tissue and organism level context which are key to a miRNA function. Also, current technology to carry out this validation is limited in both generalizability and throughput. Methodologies with greater scalability and rapidity are required to better understand the function of these important species of RNA. To this end, there is an increasing store of RNA expression level data incorporating both miRNA and mRNA, and in this chapter, we describe how to use machine learning and gene-sets to translate the knowledge of phenotype defined by mRNA to putative roles for miRNA. We outline our approach to this process and highlight how it was done for our miRNA annotation of the hallmarks of cancer using the Cancer Genome Atlas (TCGA) dataset. The concepts we present are applicable across datasets and phenotypes, and we highlight potential pitfalls and challenges that may be faced as they are used.
Subject(s)
MicroRNAs , Neoplasms , Humans , MicroRNAs/genetics , MicroRNAs/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Machine Learning , Neoplasms/genetics , Gene Expression ProfilingABSTRACT
Tumor hypoxia is associated with poor patient outcomes in estrogen receptor-α-positive (ERα+) breast cancer. Hypoxia is known to affect tumor growth by reprogramming metabolism and regulating amino acid (AA) uptake. Here, we show that the glutamine transporter, SNAT2, is the AA transporter most frequently induced by hypoxia in breast cancer, and is regulated by hypoxia both in vitro and in vivo in xenografts. SNAT2 induction in MCF7 cells was also regulated by ERα, but it became predominantly a hypoxia-inducible factor 1α (HIF-1α)-dependent gene under hypoxia. Relevant to this, binding sites for both HIF-1α and ERα overlap in SNAT2's cis-regulatory elements. In addition, the down-regulation of SNAT2 by the ER antagonist fulvestrant was reverted in hypoxia. Overexpression of SNAT2 in vitro to recapitulate the levels induced by hypoxia caused enhanced growth, particularly after ERα inhibition, in hypoxia, or when glutamine levels were low. SNAT2 up-regulation in vivo caused complete resistance to antiestrogen and, partially, anti-VEGF therapies. Finally, high SNAT2 expression levels correlated with hypoxia profiles and worse outcome in patients given antiestrogen therapies. Our findings show a switch in the regulation of SNAT2 between ERα and HIF-1α, leading to endocrine resistance in hypoxia. Development of drugs targeting SNAT2 may be of value for a subset of hormone-resistant breast cancer.
Subject(s)
Amino Acid Transport System A/metabolism , Antineoplastic Agents, Hormonal/therapeutic use , Breast Neoplasms/pathology , Cell Hypoxia , Drug Resistance, Neoplasm , Estrogen Receptor Modulators/therapeutic use , Animals , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Cell Line, Tumor , Estrogen Receptor alpha/metabolism , Female , Heterografts , Humans , Mice , Tumor MicroenvironmentABSTRACT
BACKGROUND: Epidemiological studies suggest that metformin may reduce the incidence of cancer in patients with diabetes and multiple late phase clinical trials assessing the potential of repurposing this drug are underway. Transcriptomic profiling of tumour samples is an excellent tool to understand drug bioactivity, identify candidate biomarkers and assess for mechanisms of resistance to therapy. METHODS: Thirty-six patients with untreated primary breast cancer were recruited to a window study and transcriptomic profiling of tumour samples carried out before and after metformin treatment. RESULTS: Multiple genes that regulate fatty acid oxidation were upregulated at the transcriptomic level and there was a differential change in expression between two previously identified cohorts of patients with distinct metabolic responses. Increase in expression of a mitochondrial fatty oxidation gene composite signature correlated with change in a proliferation gene signature. In vitro assays showed that, in contrast to previous studies in models of normal cells, metformin reduces fatty acid oxidation with a subsequent accumulation of intracellular triglyceride, independent of AMPK activation. CONCLUSIONS: We propose that metformin at clinical doses targets fatty acid oxidation in cancer cells with implications for patient selection and drug combinations. CLINICAL TRIAL REGISTRATION: NCT01266486.
Subject(s)
Breast Neoplasms/drug therapy , Fatty Acids/metabolism , Metformin/pharmacology , Protein Kinases/genetics , AMP-Activated Protein Kinase Kinases , Animals , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Proliferation/drug effects , Diabetes Mellitus/drug therapy , Diabetes Mellitus/genetics , Diabetes Mellitus/metabolism , Female , Gene Expression Profiling/methods , Gene Expression Regulation, Neoplastic/drug effects , Heterografts , Humans , Lipid Metabolism/drug effects , Lipid Peroxidation/drug effects , Mice , Mitochondria/drug effects , Oxidation-Reduction/drug effects , Transcriptome/drug effectsABSTRACT
Skp1-Cul1-F-box protein (SCF) ubiquitin ligases direct cell survival decisions by controlling protein ubiquitylation and degradation. Sufu (Suppressor of fused) is a central regulator of Hh (Hedgehog) signaling and acts as a tumor suppressor by maintaining the Gli (Glioma-associated oncogene homolog) transcription factors inactive. Although Sufu has a pivotal role in Hh signaling, the players involved in controlling Sufu levels and their role in tumor growth are unknown. Here, we show that Fbxl17 (F-box and leucine-rich repeat protein 17) targets Sufu for proteolysis in the nucleus. The ubiquitylation of Sufu, mediated by Fbxl17, allows the release of Gli1 from Sufu for proper Hh signal transduction. Depletion of Fbxl17 leads to defective Hh signaling associated with an impaired cancer cell proliferation and medulloblastoma tumor growth. Furthermore, we identify a mutation in Sufu, occurring in medulloblastoma of patients with Gorlin syndrome, which increases Sufu turnover through Fbxl17-mediated polyubiquitylation and leads to a sustained Hh signaling activation. In summary, our findings reveal Fbxl17 as a novel regulator of Hh pathway and highlight the perturbation of the Fbxl17-Sufu axis in the pathogenesis of medulloblastoma.
Subject(s)
F-Box Proteins/metabolism , Hedgehog Proteins/metabolism , Medulloblastoma/pathology , Protein Processing, Post-Translational , Repressor Proteins/metabolism , Animals , Cell Line , Cell Proliferation , Disease Models, Animal , Humans , Mice , Rats , Signal Transduction , UbiquitinationABSTRACT
The nuclear pore complex (NPC) machinery is emerging as an important determinant in the maintenance of genome integrity and sensitivity to DNA double-strand break (DSB)-inducing agents, such as ionising radiation (IR). In this study, using a high-throughput siRNA screen, we identified the central channel NPC protein Nup54, and concomitantly its molecular partners Nup62 and Nup58, as novel factors implicated in radiosensitivity. Nup54 depletion caused an increase in cell death by mitotic catastrophe after IR, and specifically enhanced both the duration of the G2 arrest and the radiosensitivity of cells that contained replicated DNA at the time of IR exposure. Nup54-depleted cells also exhibited increased formation of chromosome aberrations arisen from replicated DNA. Interestingly, we found that Nup54 is epistatic with the homologous recombination (HR) factor Rad51. Moreover, using specific DNA damage repair reporters, we observed a decreased HR repair activity upon Nup54 knockdown. In agreement with a role in HR repair, we also demonstrated a decreased formation of HR-linked DNA synthesis foci and sister chromatid exchanges after IR in cells depleted of Nup54. Our study reveals a novel role for Nup54 in the response to IR and the maintenance of HR-mediated genome integrity.
Subject(s)
DNA Replication , DNA/metabolism , Nuclear Pore Complex Proteins/metabolism , Recombinational DNA Repair , Cell Line, Tumor , Cell Survival/genetics , Cell Survival/radiation effects , DNA/genetics , DNA Breaks, Double-Stranded/radiation effects , HeLa Cells , Humans , MCF-7 Cells , Nuclear Pore/genetics , Nuclear Pore/metabolism , Nuclear Pore Complex Proteins/genetics , RNA Interference , Rad51 Recombinase/genetics , Rad51 Recombinase/metabolism , Radiation, Ionizing , Sister Chromatid Exchange/radiation effectsABSTRACT
Oscillations are crucial to the normal function of living organisms, across a wide variety of biological processes. In eukaryotes, oscillatory dynamics are thought to arise from interactions at the protein and RNA levels; however, the role of non-coding RNA in regulating these dynamics remains understudied. In this work, we show how non-coding RNA acting as microRNA (miRNA) sponges in a conserved miRNA - transcription factor feedback motif, can give rise to oscillatory behaviour, and how to test for this experimentally. Control of these non-coding RNA can dynamically create oscillations or stability, and we show how this behaviour predisposes to oscillations in the stochastic limit. These results, supported by emerging evidence for the role of miRNA sponges in development, point towards key roles of different species of miRNA sponges, such as circular RNA, potentially in the maintenance of yet unexplained oscillatory behaviour. These results help to provide a paradigm for understanding functional differences between the many redundant, but distinct RNA species thought to act as miRNA sponges in nature, such as long non-coding RNA, pseudogenes, competing mRNA, circular RNA, and3' UTRs.