Substantial unannotated noncoding transcripts in tumors may transcriptionally regulate cancer-related genes.

Decision letter: Major transcriptional changes observed in the Fulani, an ethnic group less susceptible to malaria

Author response: Tau polarizes an aging transcriptional signature to excitatory neurons and glia

Background: Invasive lobular carcinoma (ILC) comprises 10-15% of breast tumors and is the second most common histological type after invasive ductal carcinoma (IDC). Patients with ILC are often diagnosed at an older age and more advanced stage than those with IDC. Late recurrences and worse long-term survival suggest the need for improved approaches to treatment optimization and exploration of molecular pathways unique to ILC. Although previous reports have described comprehensive transcriptomic profiling of ILC, these were limited by small sample sizes. Furthermore, differential gene expression between ILC and IDC within genomic risk groups and molecular subtypes has yet to be explored. Here we characterize differential gene expression between ILC and IDC in a large, age-matched patient subset categorized by 70-gene signature/MammaPrint (MP) risk and 80-gene signature/BluePrint (BP) subtype. Methods: The prospective FLEX Registry (NCT03053193) includes stage I-III primary invasive breast cancer patients who receive MP/BP testing and consent to full transcriptome and clinical data collection. This sub-analysis included 450 ILC patients enrolled from 2017 to present. Compared with a random selection of IDC patients (n=450, mean age, 60 years), ILC patients were older (mean, 63 years, p<0.001). Thus, we selected an age-matched subset for differential gene expression analysis. There were few non-Luminal ILCs; thus, gene expression analyses were limited to BP Luminal tumors. A subset of 413 age-matched pairs (n=826) of ILC and IDC were used for analysis. Gene expression data were quantile normalized using R limma package, and differentially expressed genes (DEGs) were compared between groups. DEGs with an adjusted p<0.05 and log2 fold change > ± 1.0 were considered significant. Results: ILC represented 13% of FLEX cases (n=450/3562), and were 81% lymph node-negative, 99% ER+, 94% HER2-negative, and 68% MP Low Risk (LR). By BP, ILC were 99% Luminal, 1% HER2, and <1% Basal type. BP Luminal ILC were predominantly grade 2 (63%), T1 (61%), node-negative (84%), and MP LR (69%). Menopausal status, nodal status, ethnicity, BMI distribution, and frequency of type 2 diabetes mellitus were similar between ILC and IDC. However, IDC were more likely to be MP HR (46% IDC vs. 31% ILC, p<0.001) and grade 3 (15% IDC vs. 4% ILC, p<0.001). ILC were more likely to be T3 (10% ILC vs. 1% IDC, p<0.001). We found 4 DEGs common to all comparisons: all Luminal ILC vs. IDC, MP LR ILC vs. IDC, and MP HR ILC vs. IDC. ILC had lower expression of CDH1 (E-cadherin) than IDC, regardless of MP risk. Including CDH1, 6 unique genes were differentially expressed in LR ILC compared with IDC, and 21 genes were differentially expressed in HR ILC compared with IDC. Genes with increased expression in HR ILC were related to immune cell migration/chemotaxis, hormone signaling, and growth factor signaling. HR ILCs were also enriched for TGFβ signaling and angiogenesis pathway genes. Conclusions: Here we report differential clinical and molecular characteristics between ILC and IDC in a large, age-matched patient subset. Regardless of MP risk, expression of CDH1 was lower in ILC compared with IDC. Approximately one-third of ILCs were MP HR, and we report a greater number and diversity of DEGs between HR ILC and HR IDC compared with LR tumors, in particular genes related to TGFβ signaling. TGFβ pathway genes play a variety of roles in the tumor microenvironment, including induction of angiogenesis, fibroblast growth factor stimulation, and inhibition and/or exclusion of an immune response. These results suggest that therapeutic strategies targeting the TGFβ pathway may be future avenues of exploration in ILC, although further studies are warranted to characterize underlying molecular mechanisms. Citation Format: Beth-Ann Lesnikoski, Jennifer A. Crozier, Gordan Srkalovic, Patricia Robinson, Clodia Osipo, Kaylan Banda, Heather M. Kling, Josien Haan, William Audeh, FLEX Investigators Group. Differential gene expression in luminal-type invasive lobular carcinoma and invasive ductal carcinoma by MammaPrint risk stratification [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS18-03.

Virus-induced expression of interferon (IFN)-A genes is regulated by two members of the IFN regulatory factor (IRF) family, IRF-3 and IRF-7, which are activated by phosphorylation during viral infection by the IKK-related serine/threonine kinases TBK1 and IkappaB kinase epsilon (IKKepsilon). In this study, we demonstrate that three IRF-binding sites located in the virus-responsive element mediate the transcriptional activation of the IFN-A4 promoter by IRF-3. The precise arrangement of these IRF elements is required for synergistic activation of the IFN-A4 promoter following Newcastle disease virus infection or activation by TBK1 or IKKepsilon. The ordered assembly of IRF-3 multimers on the promoter also determines cooperative recruitment of IRF-3 and CREB-binding protein and differential virus-induced expression of IFN-A4 gene promoter compared with IFN-A11. Naturally occurring nucleotide substitutions disrupt two of the IRF elements in the IFN-A11 gene promoter, leading to a dramatic decrease in IRF-3 and CREB-binding protein recruitment and in IRF-3-dependent transcription. Transcription of the IFN-A4 promoter by IRF-7 is mediated by two IRF elements; promoter mutants that carry a reversed IRF element retain the ability to respond to IKKepsilon or TBK1 expression in the presence of IRF-7 but lose the capacity to respond to virus or kinase-induced IRF-3. Interestingly, IKKepsilon or TBK1 stimulates the IRF-7-mediated transcription of IFN-A11, although at a lesser extent compared with IFN-A4. Our data indicate that virus-induced expression of IFN-A genes is dictated by the organization of IRF elements within the IFN-A promoters and that the differential IFN-A gene expression, based on the IRF-3 responsiveness, is partially compensated in the presence of IRF-7 when both factors are activated by IKKepsilon or TBK1.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the third virus that caused coronavirus-related outbreaks over the past 20 years. The outbreak was first reported in December 2019 in Wuhan, China, but rapidly progressed into a pandemic of an unprecedented scale since the 1918 flu pandemic. Besides respiratory complications in patients with COVID-19, clinical characterization of severe infection cases showed several other comorbidities, including multiple organ failure, and septic shock. To better understand the systemic pathogenesis of COVID-19, we interrogated the virus's presence in the peripheral blood cells, which might provide a form of trafficking or hiding to the virus. By analyzing >2 billion sequence reads of high-throughput transcriptome sequence data from 180 samples of patients with active SARS-CoV-2 infection or healthy controls collected from 6 studies, we found evidence of traces of SARS-CoV-2 RNA in peripheral blood mononuclear cells in two samples from two independent studies. In contrast, the viral RNA was abundant in bronchoalveolar lavage specimens from the same patients. We also devised a “viral spike-to-actin” RNA normalization as a metric to compare across various samples and minimize errors caused by intersample variability in total human RNA abundance. Our observation suggests immune presentation and discounts the possibility of extensive viral infection of lymphocytes or monocytes.

Decision letter: Single-cell transcriptomic atlas of lung microvascular regeneration after targeted endothelial cell ablation

BackgroundThe heterogeneous subtypes and stages of epithelial ovarian cancer (EOC) differ in their biological features, invasiveness, and response to chemotherapy, but the transcriptional regulators causing their differences remain nebulous.MethodsIn this study, we compared high-grade serous ovarian cancers (HGSOCs) to low malignant potential or serous borderline tumors (SBTs). Our aim was to discover new regulatory factors causing distinct biological properties of HGSOCs and SBTs.ResultsIn a discovery dataset, we identified 11 differentially expressed genes (DEGs) between SBTs and HGSOCs. Their expression correctly classified 95% of 267 validation samples. Two of the DEGs, TMEM30B and TSPAN1, were significantly associated with worse overall survival in patients with HGSOC. We also identified 17 DEGs that distinguished stage II vs. III HGSOC. In these two DEG promoter sets, we identified significant enrichment of predicted transcription factor binding sites, including those of RARA, FOXF1, BHLHE41, and PITX1. Using published ChIP-seq data acquired from multiple non-ovarian cell types, we showed additional regulatory factors, including AP2-gamma/TFAP2C, FOXA1, and BHLHE40, bound at the majority of DEG promoters. Several of the factors are known to cooperate with and predict the presence of nuclear hormone receptor estrogen receptor alpha (ER-alpha). We experimentally confirmed ER-alpha and PITX1 presence at the DEGs by performing ChIP-seq analysis using the ovarian cancer cell line PEO4. Finally, RNA-seq analysis identified recurrent gene fusion events in our EOC tumor set. Some of these fusions were significantly associated with survival in HGSOC patients; however, the fusion genes are not regulated by the transcription factors identified for the DEGs.ConclusionsThese data implicate an estrogen-responsive regulatory network in the differential gene expression between ovarian cancer subtypes and stages, which includes PITX1. Importantly, the transcription factors associated with our DEG promoters are known to form the MegaTrans complex in breast cancer. This is the first study to implicate the MegaTrans complex in contributing to the distinct biological trajectories of malignant and indolent ovarian cancer subtypes.

Intestinal epithelial cells participate in the acute phase response in response to inflammation. We have shown that acute phase protein genes are induced during intestinal acute phase response, and that the CCAAT/enhancer binding protein family of transcription factors are involved. To address the role of specific C/EBP isoforms, we generated IEC-6 rat intestinal epithelial cell lines expressing different C/EBP isoforms, by retroviral infection. Overexpression of C/EBPalpha p30 and C/EBPdelta led to increases in C/EBPbeta LAP and C/EBPbeta LIP endogenous protein levels, as determined by electrophoretic mobility shift assays and Western blot. Inhibition of C/EBP activity with dominant negative C/EBPs (C/EBPbeta LIP, 3hF, 4hF) decreased glucocorticoid-, cAMP- and IL-1 responsiveness of the endogenous haptoglobin gene, while overexpression of each C/EBP isoform increased the responsiveness to these regulators. In contrast, dominant negative C/EBPs or C/EBP isoforms did not alter the expression of alpha-acid glycoprotein in response to dexamethasone and of C/EBPbeta and C/EBPdelta in response to various regulators as assessed by Northern blot. These data show that the three C/EBP isoforms are involved in the regulation of haptoglobin and that C/EBPbeta, C/EBPdelta, and alpha-acid glycoprotein expression are not induced by C/EBP isoforms in contrast to other cell types. C/EBPbeta LAP-expressing cells showed an inhibition of cell growth characterized by a delay in p27(Kip1) decrease in response to serum and a decrease in cyclin D isoforms and cyclin E protein levels. Finally, C/EBP isoforms interact with the E2F4 transcription factor. Thus, specific C/EBP isoforms are involved in the differential expression of acute phase protein genes in response to hormones and cytokines. Furthermore, C/EBP isoforms may play a role in the control of cell cycle progression.

RATIONALE. Significant sex-differences exist in asthma across the lifespan. Asthma prevalence and severity are greater in pre-pubescent boys compared to girls but greater in women after puberty. In women, asthma may improve after menopause when sex hormones wane. To address potential mechanisms underlying these changes, we compared the airway transcriptomics signatures of post-menopausal vs. premenopausal women. METHODS. We used bulk RNA seq data from bronchial epithelial cells from three different cohorts (SARP III, GEO-GSE85567, GEO-GSE201955) to perform a comprehensive analysis of differential gene expression and regulation. Data were combined into a meta-analysis, to compare pre-menopausal (n=114 cases with asthma, 48 controls) to post-menopausal women (n=52 cases with asthma, 13 controls). Differential gene expression between pre- and postmenopausal women was analyzed separately for asthma (Figure 1A) and control groups (not shown). Significant differentially expressed genes (DEGs) were identified by contrasting effect sizes (log fold-changes) between asthma and control groups using a Z-score interaction test. Accordingly, a scatter plot (Figure 1B) was created to represent whether differential gene expression between pre- and post-menopausal conditions is modified by disease status (asthma vs. control). The scatter plot also highlights that these findings are related to asthma per. se. and not to normal aging and menopausal transition. Pathway enrichment analysis was conducted using the differential expression data, followed by differential regulation analysis of the identified pathways. Statistical significance was assessed using a false discovery rate (FDR) threshold of 5%. RESULTS. Out of 15,468 genes that passed quality control, 19 genes were differentially expressed in bronchial epithelial cells from pre- and post-menopausal women with asthma (as compared to controls) (Figure 1A). Of these, 5 genes were down-regulated (ITLN1, POSTN, FCRL3, CCL22, PCSK6), and 14 genes were up-regulated (SPP1, S100A9, A2ML1, TNFRSF10C, CSF3R, OLFM1, C1QL2, NLRP3, HAL, PI3, RNASE1, TMEM178B, TNNI3, RNF175) when comparing pre- and post-menopausal women with asthma (Figure 1A). Of these 19 DEGs, 14 were differentially expressed in pre- as compared to post-menopausal women with asthma, but not in controls (Figure 1B). Comparing pre- to post-menopausal women with asthma, biologic pathways related to the innate and adaptive immune system activation and mitochondrial function (Electron transport Chain oxidative phosphorylation and ATP production) were both differentially regulated and expressed. CONCLUSIONS. This analysis highlights several DEGs and pathways that characterize asthma pathogenesis when comparing pre- and post-menopausal women, and emphasize the role of female sex hormones on the immune system response and mitochondrial function in asthma pathogenesis.

Heart failure with preserved ejection fraction (HFpEF) is a poorly understood, multi-system disease with high morbidity and mortality. To improve our understanding of its underlying biology, we used single-nucleus RNA sequencing (snRNA-seq) to characterize cell-specific gene expression patterns in human HFpEF myocardium. Septal myocardial biopsies (2-3 mg) from 30 HFpEF patients and 29 non-failing donor controls were analyzed using the 10X Genomics platform, with nuclei isolated from combined samples (6 patients/pool). Genotype-based demultiplexing was performed with souporcell, and gene expression quantified with CellRanger and CellBender. After quality control, nuclei were clustered and annotated by cell types based on specific marker genes. Differential expression (DE) by cell-type in HFpEF vs controls was performed using limma-voom and functional analysis performed using Gene Set Enrichment Analysis. Data were compared to dilated cardiomyopathy (DCM) using prior snRNA-seq in DCM vs respective controls. We successfully demultiplexed pooled myocardial biopsies, assigning >75% of droplets to individual patients. From eight pooled samples (19 HFpEF, 24 controls), we recovered 48,886 nuclei and identified 14 cell types. Cardiomyocytes (5159 differentially expressed [DE] genes, 36%) and fibroblasts (5905 DE genes, 49%) showed the most DE genes, while endothelial cells (2143), pericytes (1812), and macrophages (1405) had fewer. Enriched pathways common to multiple cell types included transcription/translation, immune activation, metabolism, and protein quality control. Of 7848 DE genes identified via pseudo-bulk snRNA-seq, 51% were DE in fibroblasts and 47% in cardiomyocytes, compared to <20% in other cell types. Unlike dilated cardiomyopathy (DCM), sub-clustering fibroblasts did not reveal an activated fibroblast population in HFpEF. Comparative analysis between HFpEF and DCM identified transcriptional differences primarily in cardiomyocytes. This study demonstrates the power of genotype-based demultiplexing for single-cell transcriptomic analyses of small endomyocardial biopsies and identifies cardiomyocytes as the principal cell type with distinct transcriptional changes in HFpEF versus DCM. These findings, coupled with differential gene expression and functional pathway analyses, illuminate HFpEF pathways and may nominate compelling targets for future mechanistic studies and therapeutic efforts for HFpEF.

Staphylococcus epidermidis forms surface-attached aggregates (biofilms) when grown in platelet concentrates (PCs). Comparative transcriptome analyses were undertaken to investigate differential gene expression of S. epidermidis biofilms grown in PCs. Two S. epidermidis strains isolated from human skin (AZ22 and AZ39) and one strain isolated from contaminated PCs (ST02) were grown in glucose-supplemented Trypticase Soy Broth (TSBg) and PCs. RNA was extracted and sequenced using Illumina HiSeq. Differential expression analysis was done using DESeq, and significantly differentially expressed genes (DEGs) were selected. DEGs were subjected to Kyoto encyclopedia of genes and genomes and Gene Ontology analyses. Differential gene expression was validated with quantitative reverse transcription-PCR. A total of 436, 442, and 384 genes were expressed in AZ22, AZ39, and ST02, respectively. DEG analysis showed that 170, 172, and 117 genes were upregulated in PCs in comparison to TSBg, whereas 120, 135, and 89 genes were downregulated (p< .05) in mature biofilms of AZ22, AZ39, and ST02, respectively. Twenty-seven DEGs were shared by all three strains. While 76 DEGs were shared by AZ22 and AZ39, only 34 and 21 DEGs were common between ST02, and AZ22 and AZ39, respectively. Significant transcriptional expression changes were observed in genes involved in platelet-bacteria interaction, biofilm formation, production of virulence factors, and resistance to antimicrobial peptides and antibiotics. Differential gene expression in S. epidermidis is triggered by the stressful PC storage environment. Upregulation of virulence and antimicrobial resistance genes could have clinical implications for transfusion patients.

Background: The airway epithelium plays a crucial role as a mucosal barrier against environmental challenges, including traffic-related air pollution and wildfire smoke – the two most common exposures in North America. By utilizing an in-vitro exposure cell culture model and transcriptomic analysis to assess the effects of diesel exhaust (DE; a model for traffic-related air pollution) and wood smoke (WS; a model for wildfires), we can gain a comprehensive understanding of the similarities and differences in how each exposure impacts the respiratory tract. Methods: Human bronchial epithelial cells (hBEC) were collected from six healthy never-smokers undergoing a research bronchoscopy. Cells were cultured, expanded, and differentiated at air-liquid interface (ALI) for &amp;gt;21 days. Differentiated hBECs were exposed to filtered air (control condition), diesel exhaust (diluted to PM2.5 = 300 μg/m3), or woodsmoke (diluted to PM2.5 = 300 μg/m3) for 2 hours using a CULTEX in-vitro exposure system. Twenty-four hours after each exposure, the cells were harvested for RNA sequencing. Total RNA was extracted, followed by PolyA mRNA enrichment, cDNA synthesis, and sequencing library generation. Paired-end 150bp Illumina NovaSeq sequencing targeting 50 million read-pairs per library was then performed. After preprocessing, we conducted differential gene expression and pathway analysis through the nf-core RNA-seq pipeline before conducting DESeq2 differential expression analysis and Gene Set Enrichment Analysis (GSEA). Results: Transcriptome analysis revealed 283 (Up:162; Down:121) differentially expressed genes (DEGs) in DE-exposed cells compared to FA exposure (false discovery rate (FDR) &amp;lt;0.05). In contrast, there were 83 (Up:24; Down:59) DEGs in WS-exposed cells compared to control. Both DE and WS exposures elicited changes in 18 common differentially expressed transcripts (Up:5; Down:13). GSEA analysis showed enrichment of 33 and 12 KEGG Pathways in the DE and WS exposed cells, respectively, based on DEGs. GSEA further revealed shared enriched pathways (including oxidative phosphorylation and chemical carcinogenesis) between the DE and WS-exposed cells. No significant differences in cytotoxicity (LDH assay) or barrier function (transepithelial electrical resistance) were observed when comparing the FA condition to the exposure groups. Conclusions: This study demonstrates the utility of a systems biology approach in uncovering distinct and shared transcriptional responses of bronchial epithelial cells to two extremely common environmental exposures. Given some public perception of WS as inconsequential, along with alternative assumptions that similar concentrations of DE and WS likely have indistinct effects, these findings highlight the importance of understanding the intricacies of complex pollution-induced changes in airway health.

Racial/ethnic disparity in cancer refers to the disproportionate incidence of and/or mortality from various cancers among population groups. In addition to differences in social, lifestyle and structural determinants of health, there is accumulating evidence for a biologic contribution to racial/ethnic disparity in cancer. To understand further the biologic mechanisms underlying racial/ethnic disparity in cancer, the analysis of differential aggregate gene expression and mutation among cancer patients of different population groups is important. Our recently published work reported differential RNA splicing as a critical mechanism underlying prostate cancer aggressiveness and drug response in African American (AA) patients. Here, we use R to analyze the Genomic Data Commons for differential aggregate gene expression (2-fold mean change, p &amp;lt; 0.001, Wilcoxon rank sum test) and TCGASpliceSeq to analyze TCGA for differential RNA splicing (20% median change, percent spliced in) between breast (BRCA, 183 AA and 761 white), colon (COAD, 56 AA and 194 white), squamous and adenocarcinoma lung (LUSC and LUAD, 34 and 57 AA and 392 and 444 white, respectively), and prostate (PRAD, 49 AA and 307 white) cancer specimens from AA and white patients. Our analysis identified 698 differentially expressed genes (DEGs) and 114 alternative RNA splicing events (ARSs) in BRCA, 107 DEGs and 57 ARSs in COAD, 25 and 117 DEGs and 115 and 53 ARSs in LUSC and LUAD, respectively, and 25 DEGs and 71 ARSs in PRAD from AA and white patients. Notably, comparing the lists of DEGs or ARSs from each cancer type with each of the other cancer types yields a minority of overlapping DEGs or ARSs among cancer types, indicating that race-related DEGs and ARSs are largely specific to cancer type. Pathway analysis of DEGs and ARSs reveals that the majority of these genes function in pathways relevant to cancer development and progression, such as programmed cell death, DNA repair, signal transduction, gene expression and metabolism. These analyses increase understanding of molecular mechanisms underlying racial/ethnic disparity in cancer. Upon further study of the function of these variants, such DEGs and ARSs have the potential to become candidates for development of new precision medicine interventions. Citation Format: Muthana Al Abo, Daniel J. George, Jennifer A. Freedman, Steven R. Patierno. Identification of differentially expressed and spliced genes between breast, colon, lung, and prostate cancer from African American and white patients [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr B050.

The large group of negative-strand RNA viruses (NSVs) comprises many important pathogens. To identify conserved patterns in host responses, we systematically compared changes in the cellular RNA levels after infection of human hepatoma cells with nine different NSVs of different virulence degrees. RNA sequencing experiments indicated that the amount of viral RNA in host cells correlates with the number of differentially expressed host cell transcripts. Time-resolved differential gene expression analysis revealed a common set of 178 RNAs that are regulated by all NSVs analyzed. A newly developed open access web application allows downloads and visualizations of all gene expression comparisons for individual viruses over time or between several viruses. Most of the genes included in the core set of commonly differentially expressed genes (DEGs) encode proteins that serve as membrane receptors, signaling proteins and regulators of transcription. They mainly function in signal transduction and control immunity, metabolism, and cell survival. One hundred sixty-five of the DEGs encode host proteins from which 47 have already been linked to the regulation of viral infections in previous studies and 89 proteins form a complex interaction network that may function as a core hub to control NSV infections.IMPORTANCEThe infection of cells with negative-strand RNA viruses leads to the differential expression of many host cell RNAs. The differential spectrum of virus-regulated RNAs reflects a large variety of events including anti-viral responses, cell remodeling, and cell damage. Here, these virus-specific differences and similarities in the regulated RNAs were measured in a highly standardized model. A newly developed app allows interested scientists a wide range of comparisons and visualizations.

Gene Expression after Hemarthrosis Differs between FVIII-Deficient Mice Treated with Recombinant FVIII or FVIII-Fc Fusion Protein