Severus: accurate detection and characterization of somatic structural variation in tumor genomes using long reads.

Most current studies rely on short-read sequencing to detect somatic structural variation (SV) in cancer genomes. Long-read sequencing offers the advantage of better mappability and long-range phasing, which results in substantial improvements in germline SV detection. However, current long-read SV detection methods do not generalize well to the analysis of somatic SVs in tumor genomes with complex rearrangements, heterogeneity, and aneuploidy. Here, we present Severus: a method for the accurate detection of different types of somatic SVs using a phased breakpoint graph approach. To benchmark various short- and long-read SV detection methods, we sequenced five tumor/normal cell line pairs with Illumina, Nanopore, and PacBio sequencing platforms; on this benchmark Severus showed the highest F1 scores (harmonic mean of the precision and recall) as compared to long-read and short-read methods. We then applied Severus to three clinical cases of pediatric cancer, demonstrating concordance with known genetic findings as well as revealing clinically relevant cryptic rearrangements missed by standard genomic panels.

Insights into the mechanisms and structure of breakage-fusion-bridge cycles in cervical cancer using long-read sequencing.

Cervical cancer is caused by human papillomavirus (HPV) infection, has few approved targeted therapeutics, and is the most common cause of cancer death in low-resource countries. We characterized 19 cervical and four head and neck cancer cell lines using long-read DNA and RNA sequencing and identified the HPV types, HPV integration sites, chromosomal alterations, and cancer driver mutations. Structural variation analysis revealed telomeric deletions associated with DNA inversions resulting from breakage-fusion-bridge (BFB) cycles. BFB is a common mechanism of chromosomal alterations in cancer, and our study applies long-read sequencing to this important chromosomal rearrangement type. Analysis of the inversion sites revealed staggered ends consistent with exonuclease digestion of the DNA after breakage. Some BFB events are complex, involving inter- or intra-chromosomal insertions or rearrangements. None of the BFB breakpoints had telomere sequences added to resolve the dicentric chromosomes, and only one BFB breakpoint showed chromothripsis. Five cell lines have a chromosomal region 11q BFB event, with YAP1-BIRC3-BIRC2 amplification. Indeed, YAP1 amplification is associated with a 10-year-earlier age of diagnosis of cervical cancer and is three times more common in African American women. This suggests that individuals with cervical cancer and YAP1-BIRC3-BIRC2 amplification, especially those of African ancestry, might benefit from targeted therapy. In summary, we uncovered valuable insights into the mechanisms and consequences of BFB cycles in cervical cancer using long-read sequencing.

Long-Term Acetylcholinesterase Depletion Alters the Levels of Key Synaptic Proteins while Maintaining Neuronal Markers in the Aging Zebrafish (Danio rerio) Brain.

INTRODUCTION: Interventions targeting cholinergic neurotransmission like acetylcholinesterase (AChE) inhibition distinguish potential mechanisms to delay age-related impairments and attenuate deficits related to neurodegenerative diseases. However, the chronic effects of these interventions are not well described. METHODS: In the current study, global levels of cholinergic, cellular, synaptic, and inflammation-mediating proteins were assessed within the context of aging and chronic reduction of AChE activity. Long-term depletion of AChE activity was induced by using a mutant zebrafish line, and they were compared with the wildtype group at young and old ages. RESULTS: Results demonstrated that AChE activity was lower in both young and old mutants, and this decrease coincided with a reduction in ACh content. Additionally, an overall age-related reduction in AChE activity and the AChE/ACh ratio was observed, and this decline was more prominent in wildtype groups. The levels of an immature neuronal marker were upregulated in mutants, while a glial marker showed an overall reduction. Mutants had preserved levels of inhibitory and presynaptic elements with aging, whereas glutamate receptor subunit levels declined. CONCLUSION: Long-term AChE activity depletion induces synaptic and cellular alterations. These data provide further insights into molecular targets and adaptive responses following the long-term reduction of AChE activity that was also targeted pharmacologically to treat neurodegenerative diseases in human subjects.

Insights into the Mechanisms and Structure of Breakage-Fusion-Bridge Cycles in Cervical Cancer using Long-Read Sequencing.

Cervical cancer is caused by human papillomavirus (HPV) infection, has few approved targeted therapeutics, and is the most common cause of cancer death in low-resource countries. We characterized 19 cervical and four head and neck cell lines using long-read DNA and RNA sequencing and identified the HPV types, HPV integration sites, chromosomal alterations, and cancer driver mutations. Structural variation analysis revealed telomeric deletions associated with DNA inversions resulting from breakage-fusion-bridge (BFB) cycles. BFB is a common mechanism of chromosomal alterations in cancer, and this is one of the first analyses of these events using long-read sequencing. Analysis of the inversion sites revealed staggered ends consistent with exonuclease digestion of the DNA after breakage. Some BFB events are complex, involving inter- or intra-chromosomal insertions or rearrangements. None of the BFB breakpoints had telomere sequences added to resolve the dicentric chromosomes and only one BFB breakpoint showed chromothripsis. Five cell lines have a Chr11q BFB event, with YAP1/BIRC2/BIRC3 gene amplification. Indeed, YAP1 amplification is associated with a 10-year earlier age of diagnosis of cervical cancer and is three times more common in African American women. This suggests that cervical cancer patients with YAP1/BIRC2/BIRC3-amplification, especially those of African American ancestry, might benefit from targeted therapy. In summary, we uncovered new insights into the mechanisms and consequences of BFB cycles in cervical cancer using long-read sequencing.

Functional analysis of co-expression networks of zebrafish ace2 reveals enrichment of pathways associated with development and disease.

Human Angiotensin I Converting Enzyme 2 (ACE2) plays an essential role in blood pressure regulation and SARS-CoV-2 entry. ACE2 has a highly conserved, one-to-one ortholog (ace2) in zebrafish, which is an important model for human diseases. However, the zebrafish ace2 expression profile has not yet been studied during early development, between genders, across different genotypes, or in disease. Moreover, a network-based meta-analysis for the extraction of functionally enriched pathways associated with differential ace2 expression is lacking in the literature. Herein, we first identified significant development-, tissue-, genotype-, and gender-specific modulations in ace2 expression via meta-analysis of zebrafish Affymetrix transcriptomics datasets (ndatasets = 107); and the correlation analysis of ace2 meta-differential expression profile revealed distinct positively and negatively correlated local functionally enriched gene networks. Moreover, we demonstrated that ace2 expression was significantly modulated under different physiological and pathological conditions related to development, tissue, gender, diet, infection, and inflammation using additional RNA-seq datasets. Our findings implicate a novel translational role for zebrafish ace2 in organ differentiation and pathologies observed in the intestines and liver.

Global miRNA expression of bone marrow mesenchymal stem/stromal cells derived from Fanconi anemia patients.

Fanconi anemia (FA) is a rare genetic disorder characterized by genomic instability, developmental defects, and bone marrow (BM) failure. Hematopoietic stem cells (HSCs) in BM interact with the mesenchymal stem/stromal cells (MSCs); and this partly sustains the tissue homeostasis. MicroRNAs (miRNAs) can play a critical role during these interactions possibly via paracrine mechanisms. This is the first study addressing the miRNA profile of FA BM-MSCs obtained before and after BM transplantation (preBMT and postBMT, respectively). Non-coding RNA expression profiling and quality control analyses were performed in Donors (n = 13), FA preBMT (n = 11), and FA postBMT (n = 6) BM-MSCs using GeneChip miRNA 2.0 Array. Six Donor-FA preBMT pairs were used to identify a differentially expressed miRNA expression signature containing 50 miRNAs, which exhibited a strong correlation with the signature obtained from unpaired samples. Five miRNAs (hsa-miR-146a-5p, hsa-miR-148b-3p, hsa-miR-187-3p, hsa-miR-196b-5p, and hsa-miR-25-3p) significantly downregulated in both the paired and unpaired analyses were used to generate the BM-MSCs' miRNA-BM mononuclear mRNA networks upon integration of a public dataset (GSE16334; studying Donor versus FA samples). Functionally enriched KEGG pathways included cellular senescence, miRNAs, and pathways in cancer. Here, we showed that hsa-miR-146a-5p and hsa-miR-874-3p were rescued upon BMT (n = 3 triplets). The decrease in miR-146a-5p was also validated using RT-qPCR and emerged as a strong candidate as a modulator of BM mRNAs in FA patients.

Increased SGK1 activity potentiates mineralocorticoid/NaCl-induced kidney injury.

Serum and glucocorticoid-regulated kinase 1 (SGK1) stimulates aldosterone-dependent renal Na+ reabsorption and modulates blood pressure. In addition, genetic ablation or pharmacological inhibition of SGK1 limits the development of kidney inflammation and fibrosis in response to excess mineralocorticoid signaling. In this work, we tested the hypothesis that a systemic increase in SGK1 activity would potentiate mineralocorticoid/salt-induced hypertension and kidney injury. To that end, we used a transgenic mouse model with increased SGK1 activity. Mineralocorticoid/salt-induced hypertension and kidney damage was induced by unilateral nephrectomy and treatment with deoxycorticosterone acetate and NaCl in the drinking water for 6 wk. Our results show that although SGK1 activation did not induce significantly higher blood pressure, it produced a mild increase in glomerular filtration rate, increased albuminuria, and exacerbated glomerular hypertrophy and fibrosis. Transcriptomic analysis showed that extracellular matrix- and immune response-related terms were enriched in the downregulated and upregulated genes, respectively, in transgenic mice. In conclusion, we propose that systemically increased SGK1 activity is a risk factor for the development of mineralocorticoid-dependent kidney injury in the context of low renal mass and independently of blood pressure.NEW & NOTEWORTHY Increased activity of the protein kinase serum and glucocorticoid-regulated kinase 1 may be a risk factor for accelerated renal damage. Serum and glucocorticoid-regulated kinase 1 expression could be a marker for the rapid progression toward chronic kidney disease and a potential therapeutic target to slow down the process.

CHRNA5 belongs to the secondary estrogen signaling network exhibiting prognostic significance in breast cancer.

PURPOSE: Cholinergic signals can be important modulators of cellular signaling in cancer. We recently have shown that knockdown of nicotinic acetylcholine receptor subunit alpha 5, CHRNA5, diminishes the proliferative potential of breast cancer cells. However, modulation of CHRNA5 expression in the context of estrogen signaling and its prognostic implications in breast cancer remained unexplored. METHODS: Meta-analyses of large breast cancer microarray cohorts were used to evaluate the association of CHRNA5 expression with estrogen (E2) treatment, estrogen receptor (ER) status and patient prognosis. The results were validated through RT-qPCR analyses of multiple E2 treated cell lines, CHRNA5 depleted MCF7 cells and across a breast cancer patient cDNA panel. We also calculated a predicted secondary (PS) score representing direct/indirect induction of gene expression by E2 based on a public dataset (GSE8597). Co-expression analysis was performed using a weighted gene co-expression network analysis (WGCNA) pipeline. Multiple other publicly available datasets such as CCLE, COSMIC and TCGA were also analyzed. RESULTS: Herein we found that CHRNA5 expression was induced by E2 in a dose- and time-dependent manner in breast cancer cell lines. ER- breast tumors exhibited higher CHRNA5 expression levels than ER+ tumors. Independent meta-analysis for survival outcome revealed that higher CHRNA5 expression was associated with a worse prognosis in untreated breast cancer patients. Furthermore, CHRNA5 and its co-expressed gene network emerged as secondarily induced targets of E2 stimulation. These targets were largely downregulated by exposure to CHRNA5 siRNA in MCF7 cells while the response of primary ESR1 targets was dependent on the direction of the PS-score. Moreover, primary and secondary target genes were uncoupled and clustered distinctly based on multiple public datasets. CONCLUSION: Our findings strongly associate increased expression of CHRNA5 and its co-expression network with secondary E2 signaling and a worse prognosis in breast cancer.

ZenoFishDb v1.1: A Database for Xenotransplantation Studies in Zebrafish.

Rapidly accumulating literature has proven feasibility of the zebrafish xenograft models in cancer research. Nevertheless, online databases for searching the current zebrafish xenograft literature are in great demand. Herein, we have developed a manually curated database, called ZenoFishDb v1.1 (https://konulab.shinyapps.io/zenofishdb), based on R Shiny platform aiming to provide searchable information on ever increasing collection of zebrafish studies for cancer cell line transplantation and patient-derived xenografts (PDXs). ZenoFishDb v1.1 user interface contains four modules: DataTable, Visualization, PDX Details, and PDX Charts. The DataTable and Visualization pages represent xenograft study details, including injected cell lines, PDX injections, molecular modifications of cell lines, zebrafish strains, as well as technical aspects of the xenotransplantation procedures in table, bar, and/or pie chart formats. The PDX Details module provides comprehensive information on the patient details in table format and can be searched and visualized. Overall, ZenoFishDb v1.1 enables researchers to effectively search, list, and visualize different technical and biological attributes of zebrafish xenotransplantation studies particularly focusing on the new trends that make use of reporters, RNA interference, overexpression, or mutant gene constructs of transplanted cancer cells, stem cells, and PDXs, as well as distinguished host modifications.

Design, synthesis and anticancer/antiestrogenic activities of novel indole-benzimidazoles.

Indole-benzimidazoles have recently gained attention due to their antiproliferative and antiestrogenic effects. However, their structural similarities and molecular mechanisms shared with selective estrogen receptor modulators (SERMs) have not yet been investigated. In this study, we synthesized novel ethylsulfonyl indole-benzimidazole derivatives by substituting the first (R1) and fifth (R2) positions of benzimidazole and indole groups, respectively. Subsequently, we performed 1H NMR, 13C NMR, and Mass spectral and in silico docking analyses, and anticancer activity screening studies of these novel indole-benzimidazoles. The antiproliferative effects of indole-benzimidazoles were found to be more similar between the estrogen (E2) responsive cell lines MCF-7 and HEPG2 in comparison to the Estrogen Receptor negative (ER-) cell line MDA-MB-231. R1:p-fluorobenzyl group members were selected as lead compounds for their potent anticancer effects and moderate structural affinity to ER. Microarray expression profiling and gene enrichment analyses (GSEA) of the selected compounds (R1:p-fluorobenzyl: 48, 49, 50, 51; R1:3,4-difluorobenzyl: 53) helped determine the similarly modulated cellular signaling pathways among derivatives. Moreover, we identified known compounds that have significantly similar gene signatures to that of 51 via queries performed in LINCS database; and further transcriptomics comparisons were made using public GEO datasets (GSE35428, GSE7765, GSE62673). Our results strongly demonstrate that these novel indole-benzimidazoles can modulate ER target gene expression as well as dioxin-mediated aryl hydrocarbon receptor and amino acid deprivation-mediated integrated stress response signaling in a dose-dependent manner.

Enhanced expression of HNF4α during intestinal epithelial differentiation is involved in the activation of ER stress.

Intestinal epithelial cells are derived from stem cells at the crypts that undergo differentiation into transit-amplifying cells, which in turn form terminally differentiated enterocytes as these cells reach the villus. Extensive alterations in both transcriptional and translational programs occur during differentiation, which can induce the activation of cellular stress responses such as ER stress-related unfolded protein response (UPR) and autophagy, particularly in the cells that are already committed to becoming absorptive cells. Using an epithelial cell model of enterocyte differentiation, we report a mechanistic study connecting enterocyte differentiation to UPR and autophagy. We report that differentiated colon epithelial cells showed increased cytosolic Ca2+ levels and activation of all three pathways of UPR: inositol-requiring enzyme 1 (IRE1), protein kinase RNA-like ER kinase, and activating transcription factor 6 (ATF6) compared to the undifferentiated cells. Enhanced UPR in the differentiated cells was accompanied by the induction of autophagy as evidenced by increased ratio of light chain 3 II/I, upregulation of Beclin-1, and downregulation of p62. We show for the first time that mechanistically, the upregulation of hepatocyte nuclear factor 4α (HNF4α) during differentiation led to increased promoter binding and transcriptional upregulation of two major proteins of UPR: X-box binding protein-1 and ATF6, implicating HNF4α as a key regulator of UPR response during differentiation. Integrating wet-lab with in silico analyses, the present study links differentiation to cellular stress responses, and highlights the importance of transcription factor signaling and cross-talk between the cellular events in the regulation of intestinal cell differentiation.

Cholinergic Receptor Nicotinic Alpha 5 (CHRNA5) RNAi is associated with cell cycle inhibition, apoptosis, DNA damage response and drug sensitivity in breast cancer.

Cholinergic Receptor Nicotinic Alpha 5 (CHRNA5) is an important susceptibility locus for nicotine addiction and lung cancer. Depletion of CHRNA5 has been associated with reduced cell viability, increased apoptosis and alterations in cellular motility in different cancers yet not in breast cancer. Herein we first showed the expression of CHRNA5 was variable and positively correlated with the fraction of total genomic alterations in breast cancer cell lines and tumors indicating its potential role in DNA damage response (DDR). Next, we demonstrated that silencing of CHRNA5 expression in MCF7 breast cancer cell line by RNAi affected expression of genes involved in cytoskeleton, TP53 signaling, DNA synthesis and repair, cell cycle, and apoptosis. The transcription profile of CHRNA5 depleted MCF7 cells showed a significant positive correlation with that of A549 lung cancer cell line while exhibiting a negative association with the CHRNA5 co-expression profile obtained from Cancer Cell Line Encylopedia (CCLE). Moreover, it exhibited high similarities with published MCF7 expression profiles obtained from exposure to TP53 inducer nutlin-3a and topoisomerase inhibitors. We then demonstrated that CHRNA5 siRNA treatment reduced cell viability and DNA synthesis indicating G1 arrest while it significantly increased apoptotic sub-G1 cell population. Accordingly, we observed lower levels of phosphorylated RB (Ser807/811) and an increased BAX/BCL2 ratio in RNAi treated MCF7 cells. We also showed that CHRNA5 RNAi transcriptome correlated negatively with DDR relevant gene expression profile in breast cancer gene expression datasets while the coexposure to topoisomerase inhibitors in the presence of CHRNA5 RNAi enhanced chemosensitivity potentially due to reduced DDR. CHRNA5 RNAi consistently lowered total CHEK1 mRNA and protein levels as well as phosphorylated CHEK1 (Ser345) in MCF7 cells. We also detected a significant positive correlation between the expression levels of CHRNA5 and CHEK1 in CCLE, TCGA and METABRIC breast cancer datasets. Our study suggests CHRNA5 RNAi is associated with cell cycle inhibition, apoptosis as well as reduced DDR and increased drug sensitivity in breast cancer yet future studies are warranted since dose- and cell line-specific differences exist in response to CHRNA5 depletion. Gene expression microarray data can be accessed from GEO database under the accession number GSE89333.

Development of a novel zebrafish xenograft model in ache mutants using liver cancer cell lines.

Acetylcholinesterase (AChE), an enzyme responsible for degradation of acetylcholine, has been identified as a prognostic marker in liver cancer. Although in vivo Ache tumorigenicity assays in mouse are present, no established liver cancer xenograft model in zebrafish using an ache mutant background exists. Herein, we developed an embryonic zebrafish xenograft model using epithelial (Hep3B) and mesenchymal (SKHep1) liver cancer cell lines in wild-type and ache sb55 sibling mutant larvae after characterization of cholinesterase expression and activity in cell lines and zebrafish larvae. The comparison of fluorescent signal reflecting tumor size at 3-days post-injection (dpi) revealed an enhanced tumorigenic potential and a reduced migration capacity in cancer cells injected into homozygous ache sb55 mutants when compared with the wild-type. Increased tumor load was confirmed using an ALU based tumor DNA quantification method modified for use in genotyped xenotransplanted zebrafish embryos. Confocal microscopy using the Huh7 cells stably expressing GFP helped identify the distribution of tumor cells in larvae. Our results imply that acetylcholine accumulation in the microenvironment directly or indirectly supports tumor growth in liver cancer. Use of this model system for drug screening studies holds potential in discovering new cholinergic targets for treatment of liver cancers.

15-Lipoxygenase-1 re-expression in colorectal cancer alters endothelial cell features through enhanced expression of TSP-1 and ICAM-1.

15-lipoxygenase-1 (15-LOX-1) oxygenates linoleic acid to 13(S)-hydroxyoctadecadienoic acid (HODE). The enzyme is widely suppressed in different cancers and its re-expression has tumor suppressive effects. 15-LOX-1 has been shown to inhibit neoangiogenesis in colorectal cancer (CRC); in the present study we confirm this phenomenon and describe the mechanistic basis. We show that re-expression of 15-LOX-1 in CRC cell lines resulted in decreased transcriptional activity of HIF1α and reduced the expression and secretion of VEGF in both normoxic and hypoxic conditions. Conditioned medium (CM) was obtained from CRC or prostate cancer cell lines re-expressing 15-LOX-1 (15-LOX-1CM). 15-LOX-1CM treated aortic rings from 6-week old C57BL/6 mice showed significantly less vessel sprouting and more organized structure of vascular network. Human umbilical vein endothelial cells (HUVECs) incubated with 15-LOX-1CM showed reduced motility, enhanced expression of intercellular cell adhesion molecule (ICAM-1) and reduced tube formation but no change in proliferation or cell-cycle distribution. HUVECs incubated with 13(S)-HODE partially phenocopied the effects of 15-LOX-1CM, i.e., showed reduced motility and enhanced expression of ICAM-1, but did not reduce tube formation, implying the importance of additional factors. Therefore, a Proteome Profiler Angiogenesis Array was carried out, which showed that Thrombospondin-1 (TSP-1), a matrix glycoprotein known to strongly inhibit neovascularization, was expressed significantly more in HUVECs incubated with 15-LOX-1CM. TSP-1 blockage in HUVECs reduced the expression of ICAM-1 and enhanced cell motility, thereby providing a mechanism for reduced angiogenesis. The anti-angiogenic effects of 15-LOX-1 through enhanced expressions of ICAM-1 and TSP-1 are novel findings and should be explored further to develop therapeutic options.