ABSTRACT
Intra-tumor hypoxia is a common feature in many solid cancers. Although transcriptional targets of hypoxia-inducible factors (HIFs) have been well characterized, alternative splicing or processing of pre-mRNA transcripts which occurs during hypoxia and subsequent HIF stabilization is much less understood. Here, we identify many HIF-dependent alternative splicing events after whole transcriptome sequencing in pancreatic cancer cells exposed to hypoxia with and without downregulation of the aryl hydrocarbon receptor nuclear translocator (ARNT), a protein required for HIFs to form a transcriptionally active dimer. We correlate the discovered hypoxia-driven events with available sequencing data from pan-cancer TCGA patient cohorts to select a narrow set of putative biologically relevant splice events for experimental validation. We validate a small set of candidate HIF-dependent alternative splicing events in multiple human gastrointestinal cancer cell lines as well as patient-derived human pancreatic cancer organoids. Lastly, we report the discovery of a HIF-dependent mechanism to produce a hypoxia-dependent, long and coding isoform of the UDP-N-acetylglucosamine transporter SLC35A3.
Subject(s)
Alternative Splicing , Gastrointestinal Neoplasms , Humans , Aryl Hydrocarbon Receptor Nuclear Translocator/genetics , Aryl Hydrocarbon Receptor Nuclear Translocator/metabolism , Cell Line, Tumor , Gastrointestinal Neoplasms/genetics , Gastrointestinal Neoplasms/metabolism , Hypoxia-Inducible Factor 1/metabolism , Transcriptome , Tumor HypoxiaABSTRACT
PURPOSE: Treatment of BRAFV600E -mutant melanomas with MAPK inhibitors (MAPKi) results in significant tumor regression, but acquired resistance is pervasive. To understand nonmutational mechanisms underlying the adaptation to MAPKi and to identify novel vulnerabilities of melanomas treated with MAPKi, we focused on the initial response phase during treatment with MAPKi. EXPERIMENTAL DESIGN: By screening proteins expressed on the cell surface of melanoma cells, we identified the fatty acid transporter CD36 as the most consistently upregulated protein upon short-term treatment with MAPKi. We further investigated the effects of MAPKi on fatty acid metabolism using in vitro and in vivo models and analyzing patients' pre- and on-treatment tumor specimens. RESULTS: Melanoma cells treated with MAPKi displayed increased levels of CD36 and of PPARα-mediated and carnitine palmitoyltransferase 1A (CPT1A)-dependent fatty acid oxidation (FAO). While CD36 is a useful marker of melanoma cells during adaptation and drug-tolerant phases, the upregulation of CD36 is not functionally involved in FAO changes that characterize MAPKi-treated cells. Increased FAO is required for BRAFV600E -mutant melanoma cells to survive under the MAPKi-induced metabolic stress prior to acquiring drug resistance. The upfront and concomitant inhibition of FAO, glycolysis, and MAPK synergistically inhibits tumor cell growth in vitro and in vivo. CONCLUSIONS: Thus, we identified a clinically relevant therapeutic approach that has the potential to improve initial responses and to delay acquired drug resistance of BRAFV600E -mutant melanoma.
Subject(s)
Adaptation, Biological , Fatty Acids/metabolism , Melanoma/genetics , Melanoma/metabolism , Mutation , Oxidation-Reduction , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins B-raf/genetics , Alleles , Animals , Biomarkers , CD36 Antigens/genetics , CD36 Antigens/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Disease Models, Animal , Drug Resistance, Neoplasm/genetics , Gene Expression Regulation, Neoplastic/drug effects , Genotype , Glycolysis , Humans , Immunophenotyping , Melanoma/pathology , Mice , Models, Biological , Neoplasm Staging , PPAR alpha/metabolism , Xenograft Model Antitumor AssaysABSTRACT
Despite the great success of small molecule inhibitors in the treatment of patients with BRAFV600E mutated melanoma, the response to these drugs remains transient and patients eventually relapse within a few months, highlighting the need to develop novel combination therapies based on the understanding of the molecular changes induced by BRAFV600E inhibitors. The acute inhibition of oncogenic signaling can rewire entire cellular signaling pathways and thereby create novel cancer cell vulnerabilities. Here, we demonstrate that inhibition of BRAFV600E oncogenic signaling in melanoma cell lines leads to destabilization of the large subunit of RNA polymerase II POLR2A (polymerase RNA II DNA-directed polypeptide A), thereby preventing its binding to the unconventional prefoldin RPB5 interactor (URI1) chaperone complex and the successful assembly of RNA polymerase II holoenzymes. Furthermore, in melanoma cell lines treated with mitogen-activated protein kinase (MAPK) inhibitors, α-amanitin, a specific and irreversible inhibitor of RNA polymerase II, induced massive apoptosis. Pre-treatment of melanoma cell lines with MAPK inhibitors significantly reduced IC50 values to α-amanitin, creating a state of collateral vulnerability similar to POLR2A hemizygous deletions. Thus, the development of melanoma specific α-amanitin antibody-drug conjugates could represent an interesting therapeutic approach for combination therapies with BRAFV600E inhibitors.
Subject(s)
Alpha-Amanitin/pharmacology , Cell Death/drug effects , Melanoma/metabolism , Nucleic Acid Synthesis Inhibitors/pharmacology , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , RNA Polymerase II/metabolism , Apoptosis/drug effects , Cell Line, Tumor , Humans , Signal Transduction/drug effectsABSTRACT
BACKGROUND: Enhancers are genomic regulatory elements conferring spatiotemporal and signal-dependent control of gene expression. Recent evidence suggests that enhancers can generate noncoding enhancer RNAs, but their (patho)biological functions remain largely elusive. METHODS: We performed chromatin immunoprecipitation-coupled sequencing of histone marks combined with RNA sequencing of left ventricular biopsies from experimental and genetic mouse models of human cardiac hypertrophy to identify transcripts revealing enhancer localization, conservation with the human genome, and hypoxia-inducible factor 1α dependence. The most promising candidate, hypoxia-inducible enhancer RNA ( HERNA)1, was further examined by investigating its capacity to modulate neighboring coding gene expression by binding to their gene promoters by using chromatin isolation by RNA purification and λN-BoxB tethering-based reporter assays. The role of HERNA1 and its neighboring genes for pathological stress-induced growth and contractile dysfunction, and the therapeutic potential of HERNA1 inhibition was studied in gapmer-mediated loss-of-function studies in vitro using human induced pluripotent stem cell-derived cardiomyocytes and various in vivo models of human pathological cardiac hypertrophy. RESULTS: HERNA1 is robustly induced on pathological stress. Production of HERNA1 is initiated by direct hypoxia-inducible factor 1α binding to a hypoxia-response element in the histoneH3-lysine27acetylation marks-enriched promoter of the enhancer and confers hypoxia responsiveness to nearby genes including synaptotagmin XVII, a member of the family of membrane-trafficking and Ca2+-sensing proteins and SMG1, encoding a phosphatidylinositol 3-kinase-related kinase. Consequently, a substrate of SMG1, ATP-dependent RNA helicase upframeshift 1, is hyperphoshorylated in a HERNA1- and SMG1-dependent manner. In vitro and in vivo inactivation of SMG1 and SYT17 revealed overlapping and distinct roles in modulating cardiac hypertrophy. Finally, in vivo administration of antisense oligonucleotides targeting HERNA1 protected mice from stress-induced pathological hypertrophy. The inhibition of HERNA1 postdisease development reversed left ventricular growth and dysfunction, resulting in increased overall survival. CONCLUSIONS: HERNA1 is a novel heart-specific noncoding RNA with key regulatory functions in modulating the growth, metabolic, and contractile gene program in disease, and reveals a molecular target amenable to therapeutic exploitation.
Subject(s)
Cardiomyopathy, Dilated/metabolism , Cardiomyopathy, Dilated/prevention & control , Cardiomyopathy, Hypertrophic/prevention & control , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Myocytes, Cardiac/metabolism , Oligonucleotides, Antisense/administration & dosage , RNA, Untranslated/metabolism , Animals , Binding Sites , Cardiomyopathy, Dilated/genetics , Cardiomyopathy, Dilated/pathology , Cardiomyopathy, Hypertrophic/genetics , Cardiomyopathy, Hypertrophic/metabolism , Cardiomyopathy, Hypertrophic/pathology , Case-Control Studies , Disease Models, Animal , HEK293 Cells , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/deficiency , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Male , Mice, Inbred C57BL , Mice, Knockout , Myocytes, Cardiac/pathology , Promoter Regions, Genetic , RNA, Untranslated/genetics , Signal Transduction , Von Hippel-Lindau Tumor Suppressor Protein/genetics , Von Hippel-Lindau Tumor Suppressor Protein/metabolismABSTRACT
Next generation sequencing protocols such as RNA-seq have made the genome wide characterization of the transcriptome a crucial part of many research projects in biology. Analyses of the resulting data provide key information on gene expression and in certain cases on exon or isoform usage. The emergence of transcript quantification software such as Salmon has enabled researchers to efficiently estimate isoform and gene expressions across the genome while tremendously reducing the necessary computational power. Although overall gene expression estimations were shown to be accurate, isoform expression quantifications appear to be a more challenging task. Low expression levels and uneven or insufficient coverage were reported as potential explanations for inconsistent estimates. Here, through the example of the ketohexokinase ( Khk) gene in mouse, we demonstrate that the use of an incorrect gene annotation can also result in erroneous isoform quantification results. Manual correction of the input Khk gene model provided a much more accurate estimation of relative Khk isoform expression when compared to quantitative PCR (qPCR measurements). In particular, removal of an unexpressed retained intron and a proper adjustment of the 5' and 3' untranslated regions both had a strong impact on the correction of erroneous estimates. Finally, we observed a better concordance in isoform quantification between datasets and sequencing strategies when relying on the newly generated Khk annotations. These results highlight the importance of accurate gene models and annotations for correct isoform quantification and reassert the need for orthogonal methods of estimation of isoform expression to confirm important findings.
ABSTRACT
Despite strong indications that increased consumption of added sugars correlates with greater risks of developing cardiometabolic syndrome (CMS) and cardiovascular disease (CVD), independent of the caloric intake, the worldwide sugar consumption remains high. In considering the negative health impact of overconsumption of dietary sugars, increased attention is recently being given to the role of the fructose component of high-sugar foods in driving CMS. The primary organs capable of metabolizing fructose include liver, small intestine, and kidneys. In these organs, fructose metabolism is initiated by ketohexokinase (KHK) isoform C of the central fructose-metabolizing enzyme KHK. Emerging data suggest that this tissue restriction of fructose metabolism can be rescinded in oxygen-deprived environments. In this review, we highlight recent progress in understanding how fructose metabolism contributes to the development of major systemic pathologies that cooperatively promote CMS and CVD, reference recent insights into microenvironmental control of fructose metabolism under stress conditions and discuss how this understanding is shaping preventive actions and therapeutic approaches.
Subject(s)
Coronary Artery Disease/metabolism , Fructose/metabolism , Hypoxia/metabolism , Liver/metabolism , Metabolic Syndrome/metabolism , Coronary Artery Disease/epidemiology , Dietary Sucrose , Epidemics , Fructokinases/metabolism , Humans , Intestine, Small/metabolism , Kidney/metabolism , Lipid Metabolism , Metabolic Syndrome/epidemiology , Protein IsoformsABSTRACT
Motivation: Next-generation sequencing is now an established method in genomics, and massive amounts of sequencing data are being generated on a regular basis. Analysis of the sequencing data is typically performed by lab-specific in-house solutions, but the agreement of results from different facilities is often small. General standards for quality control, reproducibility and documentation are missing. Results: We developed NGS-pipe, a flexible, transparent and easy-to-use framework for the design of pipelines to analyze whole-exome, whole-genome and transcriptome sequencing data. NGS-pipe facilitates the harmonization of genomic data analysis by supporting quality control, documentation, reproducibility, parallelization and easy adaptation to other NGS experiments. Availability and implementation: https://github.com/cbg-ethz/NGS-pipe. Contact: niko.beerenwinkel@bsse.ethz.ch.
Subject(s)
High-Throughput Nucleotide Sequencing/methods , Sequence Analysis, DNA/methods , Sequence Analysis, RNA/methods , Software , Gene Expression Profiling/methods , Gene Expression Profiling/standards , Genomics/methods , High-Throughput Nucleotide Sequencing/standards , Humans , Neoplasms/genetics , Reproducibility of Results , Sequence Analysis, DNA/standards , Sequence Analysis, RNA/standardsABSTRACT
MOTIVATION: Alternative splicing represents a prime mechanism of post-transcriptional gene regulation whose misregulation is associated with a broad range of human diseases. Despite the vast availability of transcriptome data from different cell types and diseases, bioinformatics-based surveys of alternative splicing patterns remain a major challenge due to limited availability of analytical tools that combine high accuracy and rapidity. RESULTS: We describe here a novel junction-centric method, jSplice, that enables de novo extraction of alternative splicing events from RNA-sequencing data with high accuracy, reliability and speed. Application to clear cell renal carcinoma (ccRCC) cell lines and 65 ccRCC patients revealed experimentally validatable alternative splicing changes and signatures able to prognosticate ccRCC outcome. In the aggregate, our results propose jSplice as a key analytic tool for the derivation of cell context-dependent alternative splicing patterns from large-scale RNA-sequencing datasets. AVAILABILITY AND IMPLEMENTATION: jSplice is a standalone Python application freely available at http://www.mhs.biol.ethz.ch/research/krek/jsplice CONTACT: wilhelm.krek@biol.ethz.ch SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Subject(s)
Alternative Splicing , Kidney Neoplasms/genetics , Software , Transcriptome , Algorithms , Humans , Reproducibility of ResultsABSTRACT
Chaperone dependency of cancer cells is an emerging trait that relates to the need of transformed cells to cope with the various stresses associated with the malignant state. URI1 (unconventional prefoldin RPB5 interactor 1) encodes a member of the prefoldin (PFD) family of molecular chaperones that acts as part of a heterohexameric PFD complex, the URI1 complex (URI1C), to promote assembly of multiprotein complexes involved in cell signaling and transcription processes. Here, we report that human colorectal cancer (CRCs) cell lines demonstrate differential dependency on URI1 and on the URI1 partner PFD STAP1 for survival, suggesting that this differential vulnerability of CRC cells is directly linked to URI1C chaperone function. Interestingly, in URI1-dependent CRC cells, URI1 deficiency is associated with non-genotoxic p53 activation and p53-dependent apoptosis. URI1-independent CRC cells do not exhibit such effects even in the context of wildtype p53. Lastly, in tumor xenografts, the conditional depletion of URI1 in URI1-dependent CRC cells was, after tumor establishment, associated with severe inhibition of subsequent tumor growth and activation of p53 target genes. Thus, a subset of CRC cells has acquired a dependency on the URI1 chaperone system for survival, providing an example of 'non-oncogene addiction' and vulnerability for therapeutic targeting.
Subject(s)
Cell Survival/physiology , Colorectal Neoplasms/pathology , Intracellular Signaling Peptides and Proteins/metabolism , Animals , Apoptosis/physiology , Cell Line, Tumor , Colorectal Neoplasms/metabolism , Heterografts , Humans , Mice , Molecular Chaperones , Repressor Proteins , Tumor Suppressor Protein p53/metabolismABSTRACT
Pathologic cardiac growth is an adaptive response of the myocardium to various forms of systemic (e.g. pressure overload) or genetically-based (e. g. mutations in genes encoding sarcomeric proteins) stress. It represents a key aspect of different types of heart disease including aortic stenosis (AS) and hypertrophic cardiomyopathy (HCM). While many of the pathophysiological and hemodynamical aspects of pathologic cardiac hypertrophy have been uncovered during the last decades, its underlying metabolic determinants are only beginning to come into focus. Here, we review the epidemiological evidence and pathological features of hypertrophic heart disease in AS and HCM and consider in this context the development of microenvironmental tissue hypoxia as a key component of the heart's growth response to pathologic stress. We particularly reflect on recent evidence illustrating how activation of hypoxia-inducible factor (HIF) drives glycolytic and fructolytic metabolic programs to maintain ATP generation and support anabolic growth of the pathologically-stressed heart. Finally we discuss how this metabolic programs, when protracted, deprive the heart of energy leading ultimately to heart failure. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel.
Subject(s)
Cardiomegaly/metabolism , Cellular Microenvironment , Fructose/metabolism , Glycolysis , Myocardium/metabolism , Adaptation, Physiological , Adenosine Triphosphate/metabolism , Animals , Cardiomegaly/epidemiology , Cardiomegaly/pathology , Cardiomegaly/physiopathology , Cell Hypoxia , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Myocardium/pathology , Risk Factors , Signal Transduction , Stress, PhysiologicalABSTRACT
Fructose is a major component of dietary sugar and its overconsumption exacerbates key pathological features of metabolic syndrome. The central fructose-metabolising enzyme is ketohexokinase (KHK), which exists in two isoforms: KHK-A and KHK-C, generated through mutually exclusive alternative splicing of KHK pre-mRNAs. KHK-C displays superior affinity for fructose compared with KHK-A and is produced primarily in the liver, thus restricting fructose metabolism almost exclusively to this organ. Here we show that myocardial hypoxia actuates fructose metabolism in human and mouse models of pathological cardiac hypertrophy through hypoxia-inducible factor 1α (HIF1α) activation of SF3B1 and SF3B1-mediated splice switching of KHK-A to KHK-C. Heart-specific depletion of SF3B1 or genetic ablation of Khk, but not Khk-A alone, in mice, suppresses pathological stress-induced fructose metabolism, growth and contractile dysfunction, thus defining signalling components and molecular underpinnings of a fructose metabolism regulatory system crucial for pathological growth.
Subject(s)
Cardiomyopathy, Hypertrophic/metabolism , Fructokinases/metabolism , Fructose/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Phosphoproteins/metabolism , Ribonucleoprotein, U2 Small Nuclear/metabolism , Alternative Splicing , Animals , Cardiomyopathy, Hypertrophic/genetics , Cardiomyopathy, Hypertrophic/pathology , Cardiomyopathy, Hypertrophic/physiopathology , Disease Models, Animal , Fructokinases/deficiency , Fructokinases/genetics , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Isoenzymes/deficiency , Isoenzymes/genetics , Isoenzymes/metabolism , Male , Metabolic Syndrome/metabolism , Mice , Phosphoproteins/deficiency , Phosphoproteins/genetics , RNA Splicing Factors , Ribonucleoprotein, U2 Small Nuclear/deficiency , Ribonucleoprotein, U2 Small Nuclear/geneticsABSTRACT
Oxygen (O2) is an essential substrate in cellular metabolism and signaling and as such is linked to the survival and normal function of metazoans. Central to the molecular mechanisms underlying O2 homeostasis are hypoxia-inducible factors (HIFs), heterodimeric transcription factors composed of O2-regulated α subunits (HIF1A/HIF-1α or EPAS1/HIF-2α), and a constitutively expressed ARNT/HIF-1ß subunit, that serve as master regulators of the adaptive response to hypoxia. HIF1A and EPAS1 have both unique and overlapping functions in the regulation of diverse cellular processes, but so far there has been no evidence linking HIF signaling to peroxisomes. In a recent study we identified a unique function of EPAS1 as promoter of pexophagy in hepatocytes. Here we summarize our findings and discuss potential mechanisms by which EPAS1 might trigger pexophagy.
Subject(s)
Autophagy/physiology , Basic Helix-Loop-Helix Transcription Factors/metabolism , Hypoxia/metabolism , Oxygen/metabolism , Animals , Humans , Mammals/metabolismABSTRACT
PURPOSE: To define robust miRNA-based molecular classifiers for human clear cell renal cell carcinoma (ccRCC) subgrouping and prognostication. EXPERIMENTAL DESIGN: Multidimensional data of over 500 clear cell renal cell carcinoma (ccRCC) patients were retrieved from The Cancer Genome Atlas (TCGA) archive. Data analysis was based on a novel computational approach that selectively considers patients with extreme expression values of miRNAs to detect survival-associated molecular signatures. RESULTS: Our in silico analysis unveiled a novel ccRCC-specific 5-miRNA (miR-10b, miR-21, miR-143, miR-183, and miR-192) signature able, when combined with information from conventional TNM staging and the age of the patient, to prognosticate ccRCC outcome more accurately than known ccRCC miRNA signatures or TNM staging alone. Furthermore, our approach revealed the existence of 6 distinct subgroups of ccRCC characterized by discrete differences in overall survival, tumor stage, and mutational spectra in key ccRCC tumor suppressor genes. It also demonstrated that BAP1 mutations correlate with tumor progression rather than overall survival. CONCLUSION: Integrated analysis of multidimensional data from the TCGA archive allowed to draw a portrait of distinct molecular subclasses of human ccRCC and to define signatures for prognosticating disease outcome. Together, these results offer new prospects for more accurate stratification and prognostication of ccRCC.
Subject(s)
Carcinoma, Renal Cell/genetics , Kidney Neoplasms/genetics , MicroRNAs/genetics , Carcinoma, Renal Cell/classification , Carcinoma, Renal Cell/pathology , Cohort Studies , Gene Expression Profiling , Genomics/methods , Humans , Kidney Neoplasms/classification , Kidney Neoplasms/pathology , MicroRNAs/analysis , Mutation , Prognosis , Survival Analysis , Tumor Suppressor Proteins/genetics , Ubiquitin Thiolesterase/geneticsABSTRACT
Peroxisomes play a central role in lipid metabolism, and their function depends on molecular oxygen. Low oxygen tension or von Hippel-Lindau (Vhl) tumor suppressor loss is known to stabilize hypoxia-inducible factors alpha (Hif-1α and Hif-2α) to mediate adaptive responses, but it remains unknown if peroxisome homeostasis and metabolism are interconnected with Hif-α signaling. By studying liver-specific Vhl, Vhl/Hif1α, and Vhl/Hif2α knockout mice, we demonstrate a regulatory function of Hif-2α signaling on peroxisomes. Hif-2α activation augments peroxisome turnover by selective autophagy (pexophagy) and thereby changes lipid composition reminiscent of peroxisomal disorders. The autophagy receptor Nbr1 localizes to peroxisomes and is likewise degraded by Hif-2α-mediated pexophagy. Furthermore, we demonstrate that peroxisome abundance is reduced in VHL-deficient human clear cell renal cell carcinomas with high HIF-2α levels. These results establish Hif-2α as a negative regulator of peroxisome abundance and metabolism and suggest a mechanism by which cells attune peroxisomal function with oxygen availability.
Subject(s)
Autophagy , Basic Helix-Loop-Helix Transcription Factors/metabolism , Peroxisomes/metabolism , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Carcinoma, Renal Cell/genetics , Carcinoma, Renal Cell/metabolism , Cell Line, Tumor , Gene Deletion , Gene Expression Regulation, Neoplastic , Humans , Kidney/metabolism , Kidney Neoplasms/genetics , Kidney Neoplasms/metabolism , Mice , Mice, Knockout , Peroxisomes/genetics , Von Hippel-Lindau Tumor Suppressor Protein/genetics , Von Hippel-Lindau Tumor Suppressor Protein/metabolismABSTRACT
Endometrial cancer is the most frequently occurring malignancy of the female genital tract in Western countries. Although in many cases surgically curable, about 30% of the tumours represent an aggressive and untreatable disease. In an attempt to establish a reliable prognostic marker for endometrial carcinomas disregarding their histological diversity, we investigated the expression of KPNA2, a mediator of nucleocytoplasmic transport, and other cell proliferation-associated proteins and their correlation with cancer progression. We analysed patient tissue microarrays (TMAs) assembled from 527 endometrial cancer tissue specimens and uterus samples from a Trp53 knockout mouse model of endometrial cancer. Our data show that KPNA2 expression was significantly up-regulated in human endometrial carcinomas and associated with higher tumour grade (p = 0.026), higher FIGO stage (p = 0.027), p53 overexpression (p < 0.001), activation of the PI3K/AKT pathway, and epithelial-mesenchymal transition. Increased nuclear KPNA2 immunoreactivity was identified as a novel predictor of overall survival, independent of well-established prognostic factors in Cox regression analyses (hazard ratio 1.7, 95% CI 1.13-2.56, p = 0.01). No significant association between KPNA2 expression and endometrial cancer subtype was detected. In the mouse model, KPNA2 showed increased expression levels from precancerous (EmgD, EIC) to far-advanced invasive lesions. We further investigated the cell proliferation capacity after siRNA-mediated KPNA2 knockdown in the human endometrial cancer cell line MFE-296. KPNA2 silencing led to decreased proliferation of the cancer cells, suggesting interplay of the protein with the cell cycle. Taken together, increased expression of KPNA2 is an independent prognostic marker for poor survival. The mechanism of enhanced nucleocytoplasmic transport by KPNA2 overexpression seems a common event in aggressive cancers since we have shown a significant correlation of KPNA2 expression and tumour aggressiveness in a large variety of other solid tumour entities. Introducing KPNA2 immunohistochemistry in routine diagnostics may allow for the identification of patients who need more aggressive treatment regimens.
Subject(s)
Cell Proliferation , Endometrial Neoplasms/metabolism , Nuclear Proteins/metabolism , alpha Karyopherins/metabolism , Animals , Biomarkers, Tumor/analysis , Cell Line, Tumor , Endometrial Neoplasms/pathology , Epithelial-Mesenchymal Transition/physiology , Female , Humans , Mice , Phosphatidylinositol 3-Kinases/metabolismABSTRACT
Phenotypic heterogeneity of cancer cells, cell biological context, heterotypic crosstalk and the microenvironment are key determinants of the multistep process of tumor development. They sign responsible, to a significant extent, for the limited response and resistance of cancer cells to molecular-targeted therapies. Better functional knowledge of the complex intra- and intercellular signaling circuits underlying communication between the different cell types populating a tumor tissue and of the systemic and local factors that shape the tumor microenvironment is therefore imperative. Sophisticated 3D multicellular tumor spheroid (MCTS) systems provide an emerging tool to model the phenotypic and cellular heterogeneity as well as microenvironmental aspects of in vivo tumor growth. In this review we discuss the cellular, chemical and physical factors contributing to zonation and cellular crosstalk within tumor masses. On this basis, we further describe 3D cell culture technologies for growth of MCTS as advanced tools for exploring molecular tumor growth determinants and facilitating drug discovery efforts. We conclude with a synopsis on technological aspects for on-line analysis and post-processing of 3D MCTS models.
Subject(s)
Cell Culture Techniques/methods , Drug Discovery/methods , Neoplasms , Spheroids, Cellular/physiology , Tumor Microenvironment/physiology , Animals , Antineoplastic Agents/administration & dosage , Cell Culture Techniques/trends , Drug Discovery/trends , Humans , Neoplasms/drug therapy , Neoplasms/pathology , Spheroids, Cellular/drug effects , Spheroids, Cellular/pathology , Tumor Microenvironment/drug effectsABSTRACT
Chromosomal instability enables tumor development, enabled in part by aberrant expression of the mitotic checkpoint protein Mad2. Here we identify a novel regulatory mechanism for Mad2 expression involving miR-28-5p-mediated inhibition of Mad2 translation, and we demonstrate that this mechanism is triggered by inactivation of the tumor suppressor VHL, the most common event in clear cell renal cell carcinoma (ccRCC). In VHL-positive cancer cells, enhanced expression of miR-28-5p diminished Mad2 levels and promoted checkpoint weakness and chromosomal instability. Conversely, in checkpoint-deficient VHL-negative renal carcinoma cells, inhibition of miR-28-5p function restored Mad2 levels, mitotic checkpoint proficiency, and chromosomal stability. Notably, chromosome missegregation errors and aneuploidy that were produced in a mouse model of acute renal injury (as a result of kidney-specific ablation of pVHL function) were reverted in vivo also by genetic inhibition of miR-28-5p. Finally, bioinformatic analyses in human ccRCC associated loss of VHL with increased miR-28-5p expression and chromosomal instability. Together, our results defined miR-28-5p as a critical regulator of Mad2 translation and mitotic checkpoint function. By identifying a potential mediator of chromosomal instability in VHL-associated cancers, our work also suggests a novel microRNA-based therapeutic strategy to target aneuploid cells in VHL-associated cancers.
Subject(s)
Chromosomal Instability , Mad2 Proteins/genetics , MicroRNAs/genetics , Peptide Chain Initiation, Translational , Von Hippel-Lindau Tumor Suppressor Protein/genetics , 3' Untranslated Regions , Aneuploidy , Animals , Carcinoma, Renal Cell/genetics , Carcinoma, Renal Cell/metabolism , Cell Cycle Checkpoints , Chromosome Segregation , Female , HCT116 Cells , HeLa Cells , Humans , Kidney Neoplasms/genetics , Kidney Neoplasms/metabolism , Mad2 Proteins/metabolism , Mice , Mice, Knockout , RNA Interference , RNA, Small Interfering/geneticsABSTRACT
Loss of primary cilia is a key feature of von Hippel-Lindau tumor suppressor (VHL)-associated pathology. Although VHL-deficiency predisposes cells to precipitous cilia disassembly in response to growth factor cues, it does not affect ciliogenesis. Here, using a siRNA-based screen to find genes that are essential for ciliogenesis only in the presence of the VHL tumor suppressor gene product pVHL, we identify ubiquitin-specific protease (USP)8. The pVHL-dependency of USP8 for ciliogenesis is directly linked to its function as a HIF1α deubiquitinating enzyme. By counteracting pVHL-mediated ubiquitination of HIF1α, USP8 maintains a basal expression of HIF1α and HIF transcriptional output in normoxia, including the repression of Rabaptin5, which is essential for endosome trafficking-mediated ciliogenesis.
Subject(s)
Cilia/physiology , Endopeptidases/physiology , Endosomal Sorting Complexes Required for Transport/physiology , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Ubiquitin Thiolesterase/physiology , Ubiquitination , Animals , Cell Hypoxia , Endocytosis , HEK293 Cells , Humans , Mice , Oxygen/metabolism , Protein Binding , Protein Stability , Protein Transport , Vesicular Transport Proteins/metabolism , Von Hippel-Lindau Tumor Suppressor Protein/genetics , Von Hippel-Lindau Tumor Suppressor Protein/metabolismABSTRACT
The von Hippel-Lindau (VHL) tumor suppressor protein pVHL is commonly mutated in clear cell renal cell carcinoma (ccRCC) and has been implicated in the control of multiple cellular processes that might be linked to tumor suppression, including promoting proper spindle orientation and chromosomal stability. However, it is unclear whether pVHL exerts these mitotic regulatory functions in vivo as well. Here, we applied ischemic kidney injury to stimulate cell division in otherwise quiescent mouse adult kidneys. We show that in the short term (5.5 days after surgery), Vhl-deficient kidney cells demonstrate both spindle misorientation and aneuploidy. The spindle misorientation phenotype encompassed changes in directed cell division, which may manifest in the development of cystic lesions, whereas the aneuploidy phenotype involved the occurrence of lagging chromosomes but not chromosome bridges, indicative of mitotic checkpoint impairment. Intriguingly, in the long term (4 months after the ischemic insult), Vhl-deficient kidneys displayed a heterogeneous pattern of ccRCC precursor lesions, including cysts, clear cell-type cells, and dysplasia. Together, these data provide direct evidence for a key role of pVHL in mediating oriented cell division and faithful mitotic checkpoint function in the renal epithelium, emphasizing the importance of pVHL as a controller of mitotic fidelity in vivo.
Subject(s)
Carcinogenesis/metabolism , Mitosis , Von Hippel-Lindau Tumor Suppressor Protein/physiology , Aneuploidy , Animals , Apoptosis , Carcinoma, Renal Cell/metabolism , Carcinoma, Renal Cell/pathology , Cell Proliferation , Cells, Cultured , Chromosomal Instability , Chromosome Segregation , Epithelial Cells/physiology , Kidney Neoplasms/metabolism , Kidney Neoplasms/pathology , Kidney Tubules/metabolism , Kidney Tubules/pathology , Mice , Mice, Knockout , Spindle Apparatus/metabolismABSTRACT
Cancer-associated mutations in oncogene products and tumor suppressors contributing to tumor progression manifest themselves, at least in part, by deregulating microtubule-dependent cellular processes that play important roles in many cell biological pathways, including intracellular transport, cell architecture, and primary cilium and mitotic spindle organization. An essential characteristic of microtubules in the performance of these varied cell processes is their ability to continuously remodel, a phenomenon known as dynamic instability. It is therefore conceivable that part of the normal function of certain cancer-causing genes is to regulate microtubule dynamic instability. Here, we report the results of a high-resolution live-cell image-based RNA interference screen targeting a collection of 70 human tumor suppressor genes to uncover cancer genes affecting microtubule dynamic instability. Extraction and computational analysis of microtubule dynamics from EB3-GFP time-lapse image sequences identified the products of the tumor suppressor genes NF1 and NF2 as potent microtubule-stabilizing proteins. Further in-depth characterization of NF2 revealed that it binds to and stabilizes microtubules through attenuation of tubulin turnover by lowering both rates of microtubule polymerization and depolymerization as well as by reducing the frequency of microtubule catastrophes. The latter function appears to be mediated, in part, by inhibition of hydrolysis of tubulin-bound GTP on the growing microtubule plus end.