Genomic and microenvironmental heterogeneity shaping epithelial-to-mesenchymal trajectories in cancer

The epithelial to mesenchymal transition (EMT) is a key cellular process underlying cancer progression, with multiple intermediate states whose molecular hallmarks remain poorly characterised. To fill this gap, we present a method to robustly evaluate EMT transformation in individual tumours based on transcriptomic signals. We apply this approach to explore EMT trajectories in 7180 tumours of epithelial origin and identify three macro-states with prognostic and therapeutic value, attributable to epithelial, hybrid E/M and mesenchymal phenotypes. We show that the hybrid state is relatively stable and linked with increased aneuploidy. We further employ spatial transcriptomics and single cell datasets to explore the spatial heterogeneity of EMT transformation and distinct interaction patterns with cytotoxic, NK cells and fibroblasts in the tumour microenvironment. Additionally, we provide a catalogue of genomic events underlying distinct evolutionary constraints on EMT transformation. This study sheds light on the aetiology of distinct stages along the EMT trajectory, and highlights broader genomic and environmental hallmarks shaping the mesenchymal transformation of primary tumours

Platelet gene expression profile in acute myocardial infarction

Acute myocardial infarction is a sudden event that is fatal in around one-third of patients. It is primarily due to coronary atherosclerotic plaque rupture with subsequent platelet (PLT) activation/aggregation and thrombus formation. PLTs have a key role in the genesis and progression of atherosclerosis and in thrombus formation1. PLTs are anucleated cells which retain mRNA from their megakaryocyte precursor, therefore PLT mRNA is unique in representing a nearly fixed transcriptome2. We tested the hypothesis that platelet transcriptome acts as a fingerprint indicating the development of a future myocardial infarction, with the final goal of identifying a specific STEMI gene-signature, able to discriminate patients with acute event from healthy donors (HD) and from patients affected by stable coronary artery disease (sCAD), the phenotypically closest clinical condition to STEMI. Peripheral blood samples (50mL) were collected in Na-citrate tubes from 20 myocardial infarction patients (MI), 20 sCAD and 20 HD. Highly purified platelets were obtained by leukocyte depletion as previously described3. Platelet RNA extraction were performed by TRIzolTM reagent according to the manufacturer’s protocol. Gene expression profile was analyzed using an Affymetrix GeneChip system (Cancer Genomics and Bioinformatics Laboratory Facility, Kimmel Cancer Center, Jefferson University. Philadelphia, US). The exploratory analysis of PLT transcriptome confirmed differences in gene expression between STEMI, sCAD and HD. Hence, the common differentially expressed genes (DEGs) derived from the STEMI vs sCAD and STEMI vs HD comparisons were obtained and tested by k-nearest neighbor classification and bootstrap. A set of 17 STEMI-related DEGs was identified, showing good sensitivity and specificity for the discrimination of STEMI patients. Overall, we described a STEMI-specific gene expression patterns, suggesting that PLT transcriptome allows to characterize a powerful fingerprint of STEMI theoretically able to predict a future acute event

Repression of Esophageal Neoplasia and Inflammatory Signaling by Anti-miR-31 Delivery In Vivo.

BACKGROUND: Overexpression of microRNA-31 (miR-31) is implicated in the pathogenesis of esophageal squamous cell carcinoma (ESCC), a deadly disease associated with dietary zinc deficiency. Using a rat model that recapitulates features of human ESCC, the mechanism whereby Zn regulates miR-31 expression to promote ESCC is examined. METHODS: To inhibit in vivo esophageal miR-31 overexpression in Zn-deficient rats (n = 12-20 per group), locked nucleic acid-modified anti-miR-31 oligonucleotides were administered over five weeks. miR-31 expression was determined by northern blotting, quantitative polymerase chain reaction, and in situ hybridization. Physiological miR-31 targets were identified by microarray analysis and verified by luciferase reporter assay. Cellular proliferation, apoptosis, and expression of inflammation genes were determined by immunoblotting, caspase assays, and immunohistochemistry. The miR-31 promoter in Zn-deficient esophagus was identified by ChIP-seq using an antibody for histone mark H3K4me3. Data were analyzed with t test and analysis of variance. All statistical tests were two-sided. RESULTS: In vivo, anti-miR-31 reduced miR-31 overexpression (P = .002) and suppressed the esophageal preneoplasia in Zn-deficient rats. At the same time, the miR-31 target Stk40 was derepressed, thereby inhibiting the STK40-NF-κΒ-controlled inflammatory pathway, with resultant decreased cellular proliferation and activated apoptosis (caspase 3/7 activities, fold change = 10.7, P = .005). This same connection between miR-31 overexpression and STK40/NF-κΒ expression was also documented in human ESCC cell lines. In Zn-deficient esophagus, the miR-31 promoter region and NF-κΒ binding site were activated. Zn replenishment restored the regulation of this genomic region and a normal esophageal phenotype. CONCLUSIONS: The data define the in vivo signaling pathway underlying interaction of Zn deficiency and miR-31 overexpression in esophageal neoplasia and provide a mechanistic rationale for miR-31 as a therapeutic target for ESCC

Genome-wide definition of promoter and enhancer usage during neural induction of human embryonic stem cells

Genome-wide mapping of transcriptional regulatory elements is an essential tool for understanding the molecular events orchestrating self-renewal, commitment and differentiation of stem cells. We combined high-throughput identification of transcription start sites with genome-wide profiling of histones modifications to map active promoters and enhancers in embryonic stem cells (ESCs) induced to neuroepithelial-like stem cells (NESCs). Our analysis showed that most promoters are active in both cell types while approximately half of the enhancers are cell-specific and account for most of the epigenetic changes occurring during neural induction, and most likely for the modulation of the promoters to generate cell-specific gene expression programs. Interestingly, the majority of the promoters activated or up-regulated during neural induction have a "bivalent" histone modification signature in ESCs, suggesting that developmentally-regulated promoters are already poised for transcription in ESCs, which are apparently pre-committed to neuroectodermal differentiation. Overall, our study provides a collection of differentially used enhancers, promoters, transcription starts sites, protein-coding and non-coding RNAs in human ESCs and ESC-derived NESCs, and a broad, genome-wide description of promoter and enhancer usage and of gene expression programs characterizing the transition from a pluripotent to a neural-restricted cell fate

Targeted Gene Addition in Human Epithelial Stem Cells by Zinc-finger Nuclease-mediated Homologous Recombination

Preclinical and clinical studies showed that autologous transplantation of epidermis derived from genetically modified epithelial stem cells (EpSCs) leads to long-term correction of inherited skin adhesion defects. These studies were based on potentially genotoxic retroviral vectors. We developed an alternative gene transfer strategy aimed at targeting a “safe harbor” locus, the adeno-associated virus integration site 1 (AAVS1), by zinc-finger nuclease (ZFN)-induced homologous recombination (HR). Delivery of AAVS1-specific ZFNs and a GFP-expressing HR cassette by integration-defective lentiviral (LV) vectors (IDLVs) or adenoviral (Ad) vectors resulted in targeted gene addition with an efficiency of >20% in a human keratinocyte cell line, >10% in immortalized keratinocytes, and <1% in primary keratinocytes. Deep sequencing of the AAVS1 locus showed that ZFN-induced double-strand breaks are mostly repaired by nonhomologous end joining (NHEJ) in primary cells, indicating that poor induction of the HR-dependent DNA repair pathway may be a significant limitation for targeted gene integration. Skin equivalents derived from unselected keratinocyte cultures coinfected with a GFP-IDLV and a ZFN-Ad vector were grafted onto immunodeficient mice. GFP-positive clones were observed in all grafts up to 18 weeks post-transplantation. By histological and molecular analysis, we were able to demonstrate highly efficient targeting of the AAVS1 locus in human repopulating EpSCs

The calcineurin-NFAT pathway controls activity-dependent circadian gene expression in slow skeletal muscle

OBJECTIVE: Physical activity and circadian rhythms are well-established determinants of human health and disease, but the relationship between muscle activity and the circadian regulation of muscle genes is a relatively new area of research. It is unknown whether muscle activity and muscle clock rhythms are coupled together, nor whether activity rhythms can drive circadian gene expression in skeletal muscle. METHODS: We compared the circadian transcriptomes of two mouse hindlimb muscles with vastly different circadian activity patterns, the continuously active slow soleus and the sporadically active fast tibialis anterior, in the presence or absence of a functional skeletal muscle clock (skeletal muscle-specific Bmal1 KO). In addition, we compared the effect of denervation on muscle circadian gene expression. RESULTS:We found that different skeletal muscles exhibit major differences in their circadian transcriptomes, yet core clock gene oscillations were essentially identical in fast and slow muscles. Furthermore, denervation caused relatively minor changes in circadian expression of most core clock genes, yet major differences in expression level, phase and amplitude of many muscle circadian genes. CONCLUSIONS: We report that activity controls the oscillation of around 15% of skeletal muscle circadian genes independently of the core muscle clock, and we have identified the Ca2+-dependent calcineurin-NFAT pathway as an important mediator of activity-dependent circadian gene expression, showing that circadian locomotor activity rhythms drive circadian rhythms of NFAT nuclear translocation and target gene expression

Genomic hallmarks and therapeutic implications of G0 cell cycle arrest in cancer

BackgroundTherapy resistance in cancer is often driven by a subpopulation of cells that are temporarily arrested in a non-proliferative G0 state, which is difficult to capture and whose mutational drivers remain largely unknown.ResultsWe develop methodology to robustly identify this state from transcriptomic signals and characterise its prevalence and genomic constraints in solid primary tumours. We show that G0 arrest preferentially emerges in the context of more stable, less mutated genomes which maintain TP53 integrity and lack the hallmarks of DNA damage repair deficiency, while presenting increased APOBEC mutagenesis. We employ machine learning to uncover novel genomic dependencies of this process and validate the role of the centrosomal gene CEP89 as a modulator of proliferation and G0 arrest capacity. Lastly, we demonstrate that G0 arrest underlies unfavourable responses to various therapies exploiting cell cycle, kinase signalling and epigenetic mechanisms in single-cell data.ConclusionsWe propose a G0 arrest transcriptional signature that is linked with therapeutic resistance and can be used to further study and clinically track this state

Genomic hallmarks and therapeutic implications of G0 cell cycle arrest in cancer

BACKGROUND: Therapy resistance in cancer is often driven by a subpopulation of cells that are temporarily arrested in a non-proliferative G0 state, which is difficult to capture and whose mutational drivers remain largely unknown. RESULTS: We develop methodology to robustly identify this state from transcriptomic signals and characterise its prevalence and genomic constraints in solid primary tumours. We show that G0 arrest preferentially emerges in the context of more stable, less mutated genomes which maintain TP53 integrity and lack the hallmarks of DNA damage repair deficiency, while presenting increased APOBEC mutagenesis. We employ machine learning to uncover novel genomic dependencies of this process and validate the role of the centrosomal gene CEP89 as a modulator of proliferation and G0 arrest capacity. Lastly, we demonstrate that G0 arrest underlies unfavourable responses to various therapies exploiting cell cycle, kinase signalling and epigenetic mechanisms in single-cell data. CONCLUSIONS: We propose a G0 arrest transcriptional signature that is linked with therapeutic resistance and can be used to further study and clinically track this state

Targeted cancer exome sequencing reveals recurrent mutations in myeloproliferative neoplasms

With the intent of dissecting the molecular complexity of Philadelphia-negative myeloproliferative neoplasms (MPN), we designed a target enrichment panel to explore, using next-generation sequencing (NGS), the mutational status of an extensive list of 2,000 cancer-associated genes and microRNAs. The genomic DNA of granulocytes and in-vitro-expanded CD3+ T-lymphocytes, as a germline control, was target-enriched and sequenced in a learning cohort of 20 MPN patients using Roche 454 technology. We identified 141 genuine somatic mutations, most of which were not previously described. To test the frequency of the identified variants, a larger validation cohort of 189 MPN patients was additionally screened for these mutations using Ion Torrent AmpliSeq NGS. Excluding the genes already described in MPN, for 8 genes (SCRIB, MIR662, BARD1, TCF12, FAT4, DAP3, POLG, and NRAS), we demonstrated a mutation frequency between 3 and 8%. We also found that mutations at codon 12 of NRAS (NRASG12V and NRASG12D) were significantly associated, for primary myelofibrosis (PMF), with highest DIPSS-plus score categories. This association was then confirmed in 66 additional PMF patients composing a final dataset of 168 PMF showing an NRAS mutation frequency of 4.7%, which was associated with a worse outcome, as defined by the DIPSS plus score

Dynamic Effects of Topoisomerase I Inhibition on R-Loops and Short Transcripts at Active Promoters.

Topoisomerase I-DNA-cleavage complexes (Top1cc) stabilized by camptothecin (CPT) have specific effects at transcriptional levels. We recently reported that Top1cc increase antisense transcript (aRNAs) levels at divergent CpG-island promoters and, transiently, DNA/RNA hybrids (R-loop) in nuclear and mitochondrial genomes of colon cancer HCT116 cells. However, the relationship between R-loops and aRNAs was not established. Here, we show that aRNAs can form R-loops in N-TERA-2 cells under physiological conditions, and that promoter-associated R-loops are somewhat increased and extended in length immediately upon cell exposure to CPT. In contrast, persistent Top1ccs reduce the majority of R-loops suggesting that CPT-accumulated aRNAs are not commonly involved in R-loops. The enhancement of aRNAs by Top1ccs is present both in human colon cancer HCT116 cells and WI38 fibroblasts suggesting a common response of cancer and normal cells. Although Top1ccs lead to DSB and DDR kinases activation, we do not detect a dependence of aRNA accumulation on ATM or DNA-PK activation. However, we showed that the cell response to persistent Top1ccs can involve an impairment of aRNA turnover rather than a higher synthesis rate. Finally, a genome-wide analysis shows that persistent Top1ccs also determine an accumulation of sense transcripts at 5'-end gene regions suggesting an increased occurrence of truncated transcripts. Taken together, the results indicate that Top1 may regulate transcription initiation by modulating RNA polymerase-generated negative supercoils, which can in turn favor R-loop formation at promoters, and that transcript accumulation at TSS is a response to persistent transcriptional stress by Top1 poisoning