Alterations in gene expression and microbiome composition upon calcium-sensing receptor deletion in the mouse esophagus.

The calcium-sensing receptor (CaSR), a G protein-coupled receptor, regulates Ca2+ concentration in plasma by regulating parathyroid hormone secretion. In other tissues, it is reported to play roles in cellular differentiation and migration and in secretion and absorption. We reported previously that CaSR can be conditionally deleted in the mouse esophagus. This conditional knockout (KO) (EsoCaSR-/-) model showed a significant reduction in the levels of adherens and tight junction proteins and had a marked buildup of bacteria on the luminal esophageal surface. To further examine the role of CaSR, we used RNA sequencing to determine gene expression profiles in esophageal epithelia of control and EsoCaSR-/-mice RNA Seq data indicated upregulation of gene sets involved in DNA replication and cell cycle in EsoCaSR-/-. This is accompanied by the downregulation of gene sets involved in the innate immune response and protein homeostasis including peptide elongation and protein trafficking. Ingenuity pathway analysis (IPA) demonstrated that these genes are mapped to important biological networks including calcium and Ras homologus A (RhoA) signaling pathways. To further explore the bacterial buildup in EsoCaSR-/- esophageal tissue, 16S sequencing of the mucosal-associated bacterial microbiome was performed. Three bacterial species, g_Rodentibacter, s_Rodentibacter_unclassified, and s_Lactobacillus_hilgardi were significantly increased in EsoCaSR-/-. Furthermore, metagenomic analysis of 16S sequences indicated that pathways related to oxidative phosphorylation and metabolism were downregulated in EsoCaSR-/- tissues. These data demonstrate that CaSR impacts major pathways of cell proliferation, differentiation, cell cycle, and innate immune response in esophageal epithelium. The disruption of these pathways causes inflammation and significant modifications of the microbiome.NEW & NOTEWORTHY Calcium-sensing receptor (CaSR) plays a significant role in maintaining the barrier function of esophageal epithelium. Using RNA sequencing, we show that conditional deletion of CaSR from mouse esophagus causes upregulation of genes involved in DNA replication and cell cycle and downregulation of genes involved in the innate immune response, protein translation, and cellular protein synthesis. Pathway analysis shows disruption of signaling pathways of calcium and actin cytoskeleton. These changes caused inflammation and esophageal dysbiosis.

Screen technical noise in single cell RNA sequencing data.

We proposed a data cleaning pipeline for single cell (SC) RNA-seq data, where we first screen genes (gene-wise screening) followed by screening cell libraries (library-wise screening). Gene-wise screening is based on the expectation that for a gene with a low technical noise, a gene's count in a library will tend to increase with the increase of library size, which was tested using negative binomial regression of gene count (as dependent variable) against library size (as independent variable). Library-wise screening is based on the expectation that across-library correlations for housekeeping (HK) genes is expected to be higher than the correlations for non-housekeeping (NHK) genes in those libraries with low technical noise. We removed those libraries, whose mean pairwise correlation for HK genes is NOT significantly higher than that for NHK genes. We successfully applied the pipeline to two large SC RNA-seq datasets. The pipeline was also developed into an R package.

Reduced mitochondrial activity in colonocytes facilitates AMPKα2-dependent inflammation.

Intestinal inflammation is associated with low levels of mucosal ATP, highlighting the importance of mitochondrial function associated with ATP production in the pathophysiology of the disease. In the inflamed colon of humans and mice, we found decreased levels of mitochondrial complex cytochrome c oxidase I/IV and lower ATP levels. Thus, we generated colonic ρ0 cells with reduced mitochondrial function linked to ATP production by selective depletion of mitochondrial DNA. In these cells, RNA sequencing revealed a substantial number of differentially expressed transcripts, among which 240 belonged to inflammatory pathways activated in human inflamed colon and TNF-α-treated cells (false discovery rate < 0.05). TNF-α treatment of colonic ρ0 cells augmented IL-8 expression by 9-fold (P < 0.01) via NF-κB compared to TNF-α-treated control. Moreover, reduced mitochondrial function facilitated TNF-α-mediated NF-κB luciferase promoter activity as a result of lowered inhibitory IκBα (nuclear factor of κ light polypeptide gene enhancer in B-cell inhibitor, α), leading to elevated NF-κB. In cells with reduced mitochondrial function, TNF-α facilitated AMPKα2 activation by 8-fold (P < 0.01), which was involved in NF-κB-dependent IL-8 expression. Last, in human and mouse colon, anti-TNF-α treatment restored reduced mitochondria-dependent inflammation. We propose that selective targeting of this novel mechanism provides new treatment opportunities for intestinal inflammation.-Heller, S., Penrose, H. M., Cable, C., Biswas, D., Nakhoul, H., Baddoo, M., Flemington, E., Crawford, S. E., Savkovic, S. D. Reduced mitochondrial activity in colonocytes facilitates AMPKα2-dependent inflammation.

High-fat diet induced leptin and Wnt expression: RNA-sequencing and pathway analysis of mouse colonic tissue and tumors.

Obesity, an immense epidemic affecting approximately half a billion adults, has doubled in prevalence in the last several decades. Epidemiological data support that obesity, due to intake of a high-fat, western diet, increases the risk of colon cancer; however, the mechanisms underlying this risk remain unclear. Here, utilizing next generation RNA sequencing, we aimed to determine the high-fat diet (HFD) mediated expression profile in mouse colon and the azoxymethane/dextran sulfate sodium model of colon cancer. Mice on HFD had significantly higher colonic inflammation, tumor burden, and a number of differentially expressed transcripts compared to mice on regular diet (RD). We identified 721 transcripts differentially expressed in mouse HFD colon that were in a shared pattern with colonic tumors (RD and HFD). Importantly, in mouse colon, HFD stimulated an expression signature strikingly similar to human colon cancer, especially those with inflammatory microsatellite instability. Furthermore, pathway analysis of these transcripts demonstrated their association with active inflammation and colon cancer signaling, with leptin and Wnt as the top two transcripts elevated in mouse HFD colon shared with tumors. Moreover, in mouse colon, HFD-stimulated tumorigenic Wnt pathway activation was further validated by upregulation of ß-catenin transcriptional targets. Finally, in human colon cancer, upregulation of leptin pathway members was shown with a large network of dysregulated transcripts being linked with worse overall survival.

Latent Expression of the Epstein-Barr Virus (EBV)-Encoded Major Histocompatibility Complex Class I TAP Inhibitor, BNLF2a, in EBV-Positive Gastric Carcinomas.

The Epstein-Barr virus (EBV) BNLF2a gene product provides immune evasion properties to infected cells through inhibition of transporter associated with antigen processing (TAP)-mediated transport of antigen peptides. Although BNLF2a is considered to be a lytic gene, we demonstrate that it is expressed in nearly half of the EBV-associated gastric carcinomas analyzed. Further, we show that BNLF2a expression is dissociated from lytic gene expression. BNLF2a is therefore expressed in this latency setting, potentially helping protect the infected tumor cells from immunosurveillance.

N-linked fucosylated glycans are biomarkers for prostate cancer with a neuroendocrine and metastatic phenotype.

Prostate cancer (PCa) is a heterogeneous disease with a spectrum of pathology and outcomes ranging from indolent to lethal. Although there have been recent advancements in prognostic tissue biomarkers, limitations still exist. We leveraged Matrix Assisted Laser Desorption Ionization (MALDI) imaging of formalin-fixed, paraffin embedded (FFPE) prostate cancer specimens to determine if N-linked glycans expressed in the extracellular matrix of lethal neuroendocrine prostate cancer were also expressed in conventional prostate adenocarcinomas that were associated with poor outcomes. We found that N-glycan fucosylation was abundant in neuroendocrine prostate cancer as well as adenocarcinomas at time of prostatectomy that eventually developed recurrent metastatic disease. Analysis of patient derived xenografts revealed that this fucosylation signature was enriched differently across metastatic disease organ sites, with the highest abundance in liver metastases. These data suggest that N-linked fucosylated glycans could be an early tissue biomarker for poor PCa outcomes. Implications: These studies identify that hyper-fucosylated N-linked glycans are enriched in neuroendocrine prostate cancer and conventional prostate adenocarcinomas that progress to metastatic disease, thus advancing biomarker discovery and providing insights into mechanisms underlying metastatic disease.

Ion transport mechanisms linked to bicarbonate secretion in the esophageal submucosal glands.

The esophageal submucosal glands (SMG) secrete HCO(3)(-) and mucus into the esophageal lumen, where they contribute to acid clearance and epithelial protection. This study characterized the ion transport mechanisms linked to HCO(3)(-) secretion in SMG. We localized ion transporters using immunofluorescence, and we examined their expression by RT-PCR and in situ hybridization. We measured HCO(3)(-) secretion by using pH stat and the isolated perfused esophagus. Using double labeling with Na(+)-K(+)-ATPase as a marker, we localized Na(+)-coupled bicarbonate transporter (NBCe1) and Cl(-)-HCO(3)(-) exchanger (SLC4A2/AE2) to the basolateral membrane of duct cells. Expression of cystic fibrosis transmembrane regulator channel (CFTR) was confirmed by immunofluorescence, RT-PCR, and in situ hybridization. We identified anion exchanger SLC26A6 at the ducts' luminal membrane and Na(+)-K(+)-2Cl(-) (NKCC1) at the basolateral membrane of mucous and duct cells. pH stat experiments showed that elevations in cAMP induced by forskolin or IBMX increased HCO(3)(-) secretion. Genistein, an activator of CFTR, which does not increase intracellular cAMP, also stimulated HCO(3)(-) secretion, whereas glibenclamide, a Cl(-) channel blocker, and bumetanide, a Na(+)-K(+)-2Cl(-) blocker, decreased it. CFTR(inh)-172, a specific CFTR channel blocker, inhibited basal HCO(3)(-) secretion as well as stimulation of HCO(3)(-) secretion by IBMX. This is the first report on the presence of CFTR channels in the esophagus. The role of CFTR in manifestations of esophageal disease in cystic fibrosis patients remains to be determined.

Ulcerative colitis immune cell landscapes and differentially expressed gene signatures determine novel regulators and predict clinical response to biologic therapy.

The heterogeneous pathobiology underlying Ulcerative Colitis (UC) is not fully understood. Using publicly available transcriptomes from adult UC patients, we identified the immune cell landscape, molecular pathways, and differentially expressed genes (DEGs) across patient cohorts and their association with treatment outcomes. The global immune cell landscape of UC tissue included increased neutrophils, T CD4 memory activated cells, active dendritic cells (DC), and M0 macrophages, as well as reduced trends in T CD8, Tregs, B memory, resting DC, and M2 macrophages. Pathway analysis of DEGs across UC cohorts demonstrated activated bacterial, inflammatory, growth, and cellular signaling. We identified a specific transcriptional signature of one hundred DEGs (UC100) that distinctly separated UC inflamed from uninflamed transcriptomes. Several UC100 DEGs, with unidentified roles in UC, were validated in primary tissue. Additionally, non-responders to anti-TNFα and anti-α4ß7 therapy displayed distinct profiles of immune cells and pathways pertaining to inflammation, growth, and metabolism. We identified twenty resistant DEGs in UC non-responders to both therapies of which four had significant predictive power to treatment outcome. We demonstrated the global immune landscape and pathways in UC tissue, highlighting a unique UC signature across cohorts and a UC resistant signature with predictive performance to biologic therapy outcome.

Bacterial TLR4 and NOD2 signaling linked to reduced mitochondrial energy function in active inflammatory bowel disease.

Inflammatory bowel disease (IBD) has been linked to active signaling with bacterial components and reduced mitochondrial ATP production; however, synergism between both of these disease characteristics remains unclear. We aimed to determine in human IBD transcriptomes the link between a transcriptional signature unique to intestinal cells (ICs) with reduced mitochondrial ATP production (Mito-0) and bacteria triggered signaling using a bioinformatics approach. We generated an IC Mito-0 panel comprised of 199 differentially expressed (DE) transcripts mediated by reduced mitochondrial ATP function (DEGseq, log2 fold-change > |2|, p < .001). Transcripts from this panel were involved in diverse biological functions including regulation of mitochondrial energy (lower ATP), extracellular matrix, cell-cell contact, cytoskeleton, growth, metabolism, and inflammation. Next, unsupervised hierarchical clustering showed that the Mito-0 panel distinctly separated inflamed IBD from non-inflamed transcriptomes, which was also supported by principal component analysis (PCA) revealing distinct variation between sample types based on presence of the Mito-0 signature (PCA, p = 8.77e-09). Utilizing three independent IBD cohorts, we validated that 60 novel transcripts from the Mito-0 panel were significantly increased in inflamed tissue. Subsequently, KEGG generated bacterial TLR4 and NOD2 transcriptional signatures strongly associated with inflamed IBD transcriptomes and with the Mito-0 signature as determined by Spearman's analysis (coefficient of correlation, r = 0.92, p < .05). Herein, using a comprehensive analysis we demonstrated existence of an axis between bacteria triggered signaling and reduced mitochondrial energy function. Furthermore, we identified and validated novel transcripts within this axis as potential drivers and therapeutic targets for human IBD.

High-Throughput Sequence Analysis of Peripheral T-Cell Lymphomas Indicates Subtype-Specific Viral Gene Expression Patterns and Immune Cell Microenvironments.

Certain peripheral T-cell lymphomas (PTCLs) have been associated with viral infection, particularly infection with Epstein-Barr virus (EBV). However, a comprehensive virome analysis across PTCLs has not previously been reported. Here we utilized published whole-transcriptome RNA sequencing (RNA-seq) data sets from seven different PTCL studies and new RNA-seq data from our laboratory to screen for virus association, to analyze viral gene expression, and to assess B- and T-cell receptor diversity paradigms across PTCL subtypes. In addition to identifying EBV in angioimmunoblastic T-cell lymphoma (AITL) and extranodal NK/T-cell lymphoma (ENKTL), two PTCL subtypes with well-established EBV associations, we also detected EBV in several cases of anaplastic large-cell lymphoma (ALCL), and we found evidence of infection by the oncogenic viruses Kaposi's sarcoma-associated herpesvirus and human T-cell leukemia virus type 1 in isolated PTCL cases. In AITLs, EBV gene expression analysis showed expression of immediate early, early, and late lytic genes, suggesting either low-level lytic gene expression or productive infection in a subset of EBV-infected B-lymphocyte stromal cells. Deconvolution of immune cell subpopulations demonstrated a greater B-cell signal in AITLs than in other PTCL subtypes, consistent with a larger role for B-cell support in the pathogenesis of AITL. Reconstructed T-cell receptor (TCR) and B-cell receptor (BCR) repertoires demonstrated increased BCR diversity in AITLs, consistent with a possible EBV-driven polyclonal response. These findings indicate potential alternative roles for EBV in PTCLs, in addition to the canonical oncogenic mechanisms associated with EBV latent infection. Our findings also suggest the involvement of other viruses in PTCL pathogenesis and demonstrate immunological alterations associated with these cancers.IMPORTANCE In this study, we utilized next-generation sequencing data from 7 different studies of peripheral T-cell lymphoma (PTCL) patient samples to globally assess viral associations, provide insights into the contributions of EBV gene expression to the tumor phenotype, and assess the unique roles of EBV in modulating the immune cell tumor microenvironment. These studies revealed potential roles for EBV replication genes in some PTCL subtypes, the possible role of additional human tumor viruses in rare cases of PTCLs, and a role for EBV in providing a unique immune microenvironmental niche in one subtype of PTCLs. Together, these studies provide new insights into the understudied role of tumor viruses in PTCLs.

Loss of Forkhead Box O3 Facilitates Inflammatory Colon Cancer: Transcriptome Profiling of the Immune Landscape and Novel Targets.

BACKGROUND & AIMS: Diminished forkhead box O3 (FOXO3) function drives inflammation and cancer growth; however, mechanisms fostering these pathobiologies are unclear. Here, we aimed to identify in colon loss of FOXO3-dependent cellular and molecular changes that facilitate inflammation-mediated tumor growth. METHODS: FOXO3 knockout (KO) and wild-type (WT) mice were used in the AOM/DSS model of inflammation-mediated colon cancer. Bioinformatics were used for profiling of mRNA sequencing data from human and mouse colon and tumors; specific targets were validated in human colon cancer cells (shFOXO3). RESULTS: In mice, FOXO3 deficiency led to significantly elevated colonic tumor burden (incidence and size) compared with WT (P < .05). In FOXO3 KO colon, activated molecular pathways overlapped with those associated with mouse and human colonic inflammation and cancer, especially human colonic tumors with inflammatory microsatellite instability (false discovery rate < 0.05). FOXO3 KO colon, similar to tumors, had increased neutrophils, macrophages, B cells, T cells, and decreased natural killer cells (false discovery rate < 0.05). Moreover, in KO colon differentially expressed transcripts were linked to activation of inflammatory nuclear factor kappa B, tumorigenic cMyc, and bacterial Toll-like receptor signaling. Among differentially expressed transcripts, we validated altered expression of integrin subunit alpha 2 (ITGA2), ADAM metallopeptidase with thrombospondin type 1 motif 12, and ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 5 in mouse WT and FOXO3 KO colon and tumors (P < .05). Similarly, their altered expression was found in human inflammatory bowel disease and colon cancer tissues and linked to poor patient survival. Ultimately, in human colon cancer cells, FOXO3 knockdown (shFOXO3) led to significantly increased ITGA2, and silencing ITGA2 (siRNA) alone diminished cell growth. CONCLUSIONS: We identified the loss of FOXO3-mediated immune landscape, pathways, and transcripts that could serve as biomarkers and new targets for inflammatory colon cancer treatment.

In colonic ρ0 (rho0) cells reduced mitochondrial function mediates transcriptomic alterations associated with cancer.

BACKGROUND: Mitochondrial reprogramming has emerged as a hallmark of cancer pathobiology. Although it is believed this reprogramming is essential for cancer cells to thrive, how it supports cancer pathobiology is unclear. We previously generated colonic ρ0 (rho0) cells with reduced mitochondrial energy function and acquired their transcriptional signature. Here, we utilized a bioinformatics approach to identify their changes linked to cancer pathobiology. METHODS: Human colon cancer HCT116 cells, control and ρ0, were used for qPCR. Bioinformatics analysis: GeneCards, Kaplan-Meier Survival, GENT, cBioPortal. RESULTS: The colonic ρ0 transcriptome was linked with proliferation, DNA replication, survival, tumor morphology, and cancer. Among differentially expressed transcripts, 281 were regulators or biomarkers of human colon cancer especially those with inflammatory microsatellite instability (MSI). We identified and validated novel transcripts in ρ0 cells with altered expression in human colon cancer. Among them DGK1, HTR7, FLRT3, and ZBTB18 co-occurred with established regulators of human colon cancer pathobiology. Also, increased levels of DGKI, FLRT3, ZBTB18, and YPEL1 as well as decreased levels of HTR7, and CALML6 were linked to substantially poorer patient survival. CONCLUSION: We identified established and novel regulators in colon cancer pathobiology that are dependent on mitochondrial energy reprogramming and linked to poorer patient survival.

Ribosomopathies: mechanisms of disease.

Ribosomopathies are diseases caused by alterations in the structure or function of ribosomal components. Progress in our understanding of the role of the ribosome in translational and transcriptional regulation has clarified the mechanisms of the ribosomopathies and the relationship between ribosomal dysfunction and other diseases, especially cancer. This review aims to discuss these topics with updated information.