Comprehensive functional interrogation of susceptibility loci in GWASs identified KIAA0391 as a novel oncogenic driver via regulating pyroptosis in NSCLC.

Approximately 51 non-small-cell lung cancer (NSCLC) risk loci have been identified by genome-wide association studies (GWASs). We conducted a high throughput RNA-interference (RNAi) screening to identify the candidate causal genes in NSCLC risk loci. KIAA0391 at 14q13.1 had the highest score and could promote proliferation and metastasis of NSCLC in vitro and in vivo. We next prioritized rs3783313 as a causal variant at 14q13.1, by integrating a large-scale population study consisting of 27,120 lung cancer cases and 27,355 controls, functional annotation, and expression quantitative trait locus (eQTL) analysis. Then we found that rs3783313 could facilitate a promoter-enhancer interaction to upregulate KIAA0391 expression by affecting the affinity of transcription factor NFYA. Mechanistically, KIAA0391 knockdown dramatically influenced pyroptosis-related pathways and increased the expression of CASP1. And KIAA0391 transcriptionally repressed CASP1 by binding to SMAD2 and induced an anti-pyroptosis phenotype, promoting tumorigenesis of NSCLC, which provides new insights and potential target for NSCLC.

A new strategy for screening novel functional genes involved in reduction of lipid droplet accumulation.

Lipid droplets (LDs) are organelles that store excess lipids and provide fatty acids for energy production during starvation. LDs are also essential for cellular maintenance, but excessive accumulation of LDs triggers various cancers in addition to metabolic diseases such as diabetes. In this study, we aimed to develop a strategy to identify new genes that reduces accumulation of LDs in cancer cells using an RNA interference (RNAi) screening system employing artificial sequence-enriched shRNA libraries. Monitoring LDs by fluorescent activated cell sorting, the subsequently collected cumulative LDs cells, and shRNA sequence analysis identified a clone that potentially functioned to accumulate LDs. The clone showed no identical sequence to human Refseq. It showed very similar sequence to seven genes by allowing three mismatches. Among these genes, we identified the mediator complex subunit 6 (MED6) gene as a target of this shRNA. Silencing of MED6 led to an increase in LD accumulation and expression of the marker genes, PLIN2 and DGAT1, in fatty cells. MED6 is a member of the mediator complex that regulates RNA polymerase II transcription through transcription factor II. Some mediator complexes play important roles in both normal and pathophysiological transcription processes. These results suggest that MED6 transcriptionally regulates the genes involved in lipid metabolism and suppresses LD accumulation.

Novel kinetoplastid-specific cAMP binding proteins identified by RNAi screening for cAMP resistance in Trypanosoma brucei.

Cyclic AMP signalling in trypanosomes differs from most eukaryotes due to absence of known cAMP effectors and cAMP independence of PKA. We have previously identified four genes from a genome-wide RNAi screen for resistance to the cAMP phosphodiesterase (PDE) inhibitor NPD-001. The genes were named cAMP Response Protein (CARP) 1 through 4. Here, we report an additional six CARP candidate genes from the original sample, after deep sequencing of the RNA interference target pool retrieved after NPD-001 selection (RIT-seq). The resistance phenotypes were confirmed by individual RNAi knockdown. Highest level of resistance to NPD-001, approximately 17-fold, was seen for knockdown of CARP7 (Tb927.7.4510). CARP1 and CARP11 contain predicted cyclic AMP binding domains and bind cAMP as evidenced by capture and competition on immobilised cAMP. CARP orthologues are strongly enriched in kinetoplastid species, and CARP3 and CARP11 are unique to Trypanosoma. Localization data and/or domain architecture of all CARPs predict association with the T. brucei flagellum. This suggests a crucial role of cAMP in flagellar function, in line with the cell division phenotype caused by high cAMP and the known role of the flagellum for cytokinesis. The CARP collection is a resource for discovery of unusual cAMP pathways and flagellar biology.

ZetaSuite: computational analysis of two-dimensional high-throughput data from multi-target screens and single-cell transcriptomics.

Two-dimensional high-throughput data have become increasingly common in functional genomics studies, which raises new challenges in data analysis. Here, we introduce a new statistic called Zeta, initially developed to identify global splicing regulators from a two-dimensional RNAi screen, a high-throughput screen coupled with high-throughput functional readouts, and ZetaSuite, a software package to facilitate general application of the Zeta statistics. We compare our approach with existing methods using multiple benchmarked datasets and then demonstrate the broad utility of ZetaSuite in processing public data from large-scale cancer dependency screens and single-cell transcriptomics studies to elucidate novel biological insights.

Single B Cell Gene Co-Expression Networks Implicated in Prognosis, Proliferation, and Therapeutic Responses in Non-Small Cell Lung Cancer Bulk Tumors.

In NSCLC, there is a pressing need for immunotherapy predictive biomarkers. The processes underlying B-cell dysfunction, as well as their prognostic importance in NSCLC, are unknown. Tumor-specific B-cell gene co-expression networks were constructed by comparing the Boolean implication modeling of single-cell RNA sequencing of NSCLC tumor B cells and normal B cells. Proliferation genes were selected from the networks using in vitro CRISPR-Cas9/RNA interfering (RNAi) screening data in more than 92 human NSCLC epithelial cell lines. The prognostic and predictive evaluation was performed using public NSCLC transcriptome and proteome profiles. A B cell proliferation and prognostic gene co-expression network was present only in normal lung B cells and missing in NSCLC tumor B cells. A nine-gene signature was identified from this B cell network that provided accurate prognostic stratification using bulk NSCLC tumor transcriptome (n = 1313) and proteome profiles (n = 103). Multiple genes (HLA-DRA, HLA-DRB1, OAS1, and CD74) differentially expressed in NSCLC B cells, peripheral blood lymphocytes, and tumor T cells had concordant prognostic indications at the mRNA and protein expression levels. The selected genes were associated with drug sensitivity/resistance to 10 commonly used NSCLC therapeutic regimens. Lestaurtinib was discovered as a potential repositioning drug for treating NSCLC.

Reprogramming Mycobacterium tuberculosis CRISPR System for Gene Editing and Genome-wide RNA Interference Screening.

Mycobacterium tuberculosis is the causative agent of tuberculosis (TB), which is still the leading cause of mortality from a single infectious disease worldwide. The development of novel anti-TB drugs and vaccines is severely hampered by the complicated and time-consuming genetic manipulation techniques for M. tuberculosis. Here, we harnessed an endogenous type III-A CRISPR/Cas10 system of M. tuberculosis for efficient gene editing and RNA interference (RNAi). This simple and easy method only needs to transform a single mini-CRISPR array plasmid, thus avoiding the introduction of exogenous protein and minimizing proteotoxicity. We demonstrated that M. tuberculosis genes can be efficiently and specifically knocked in/out by this system as confirmed by DNA high-throughput sequencing. This system was further applied to single- and multiple-gene RNAi. Moreover, we successfully performed genome-wide RNAi screening to identify M. tuberculosis genes regulating in vitro and intracellular growth. This system can be extensively used for exploring the functional genomics of M. tuberculosis and facilitate the development of novel anti-TB drugs and vaccines.

Genome-Wide RNAi Screening Identifies Novel Pathways/Genes Involved in Oxidative Stress and Repurposable Drugs to Preserve Cystic Fibrosis Airway Epithelial Cell Integrity.

Recurrent infection-inflammation cycles in cystic fibrosis (CF) patients generate a highly oxidative environment, leading to progressive destruction of the airway epithelia. The identification of novel modifier genes involved in oxidative stress susceptibility in the CF airways might contribute to devise new therapeutic approaches. We performed an unbiased genome-wide RNAi screen using a randomized siRNA library to identify oxidative stress modulators in CF airway epithelial cells. We monitored changes in cell viability after a lethal dose of hydrogen peroxide. Local similarity and protein-protein interaction network analyses uncovered siRNA target genes/pathways involved in oxidative stress. Further mining against public drug databases allowed identifying and validating commercially available drugs conferring oxidative stress resistance. Accordingly, a catalog of 167 siRNAs able to confer oxidative stress resistance in CF submucosal gland cells targeted 444 host genes and multiple circuitries involved in oxidative stress. The most significant processes were related to alternative splicing and cell communication, motility, and remodeling (impacting cilia structure/function, and cell guidance complexes). Other relevant pathways included DNA repair and PI3K/AKT/mTOR signaling. The mTOR inhibitor everolimus, the α1-adrenergic receptor antagonist doxazosin, and the Syk inhibitor fostamatinib significantly increased the viability of CF submucosal gland cells under strong oxidative stress pressure. Thus, novel therapeutic strategies to preserve airway cell integrity from the harsh oxidative milieu of CF airways could stem from a deep understanding of the complex consequences of oxidative stress at the molecular level, followed by a rational repurposing of existing "protective" drugs. This approach could also prove useful to other respiratory pathologies.

Designed Antitumor Peptide for Targeted siRNA Delivery into Cancer Spheroids.

Antimicrobial/anticancer peptides (AMPs/ACPs) have shown promising results as new therapeutic agents in cancer thearpy. Among them, the designed amphiphilic α-helical peptide G(IIKK)3I-NH2 (G3) displayed great affinity and specificity in targeting cancer cells. Here, we report new insights on how G3 penetrates cancer cells. G3 showed high specificity to HCT-116 colon cancer cells compared to the HDFs (human neonatal primary dermal fibroblasts) control. With high concentrations of peptide, a clear cancer cell membrane disruption was observed through SEM. Gene knockdown of the endocytic pathways demonstrated that an energy-dependent endocytic pathway is required for the uptake of the peptide. In addition, G3 can protect and selectively deliver siRNAs into cancer cells and successfully modulated their gene expression. Gene delivery was also tested in 3D cancer spheroids and showed deep penetration delivery into the cancer spheroids. Finally, the in vivo toxicity of G3 was evaluated on zebrafish embryos, showing an increasing toxicity effect with concentration. However, the toxicity of the peptide was attenuated when complexed with siRNA. In addition, negligible toxicity was observed at the concentration range for efficient gene delivery. The current results demonstrate that G3 is promising as an excellent agent for cancer therapy.

PI3K and MAPK Pathways as Targets for Combination with the Pan-HER Irreversible Inhibitor Neratinib in HER2-Positive Breast Cancer and TNBC by Kinome RNAi Screening.

Human epidermal growth factor receptor (EGFR) 2 (HER2) is overexpressed/amplified in about 25% of all breast cancers, and EGFR is overexpressed in up to 76% and amplified in up to 24% of triple-negative breast cancers (TNBC). Here, we aimed to identify inhibitors that may enhance the anti-tumor activity of neratinib for HER2+ breast cancer and TNBC. By conducting a non-biased high-throughput RNA interference screening, we identified PI3K/AKT/mTOR and MAPK as two potential inhibitory synergistic canonical pathways. We confirmed that everolimus (mTOR inhibitor) and trametinib (MEK inhibitor) enhances combinatorial anti-proliferative effects with neratinib under anchorage-independent growth conditions (p < 0.05). Compared to single agent neratinib, the combination therapies significantly enhanced tumor growth inhibition in both SUM190 HER2+ breast cancer (neratinib plus everolimus, 77%; neratinib plus trametinib, 77%; p < 0.0001) and SUM149 TNBC (neratinib plus everolimus, 71%; neratinib plus trametinib, 81%; p < 0.0001) xenograft models. Compared to single-agent neratinib, everolimus, or trametinib, both everolimus plus neratinib and trametinib plus neratinib significantly suppressed proliferation marker Ki67 and enhanced antitumor efficacy by activating the apoptosis pathway shown by increased Bim and cleaved-PARP expression. Taken together, our data justify new neratinib-based combinations for both HER2+ breast cancer and TNBC.

SchistoCyte Atlas: A Single-Cell Transcriptome Resource for Adult Schistosomes.

Schistosomes cause untold disease and disability in the developing world. Here, we introduce SchistoCyte Atlas, a web-based platform for exploring gene expression at single-cell resolution in adult Schistosoma mansoni. Similar resources accessible to non-specialists across the globe will expedite our ability to understand the biology of these devastating parasites.

Use of circular RNAs as markers of readthrough transcription to identify factors regulating cleavage/polyadenylation events.

Circular RNAs with covalently linked ends are generated from many eukaryotic protein-coding genes when the pre-mRNA splicing machinery backsplices. These mature transcripts are resistant to digestion by exonucleases and typically have much longer half-lives than their associated linear mRNAs. Circular RNAs thus have great promise as sensitive biomarkers, including for detection of transcriptional activity. Here, we show that circular RNAs can serve as markers of readthrough transcription events in Drosophila and human cells, thereby revealing mechanistic insights into RNA polymerase II transcription termination as well as pre-mRNA 3' end processing. We describe methods that take advantage of plasmids that generate a circular RNA when an upstream polyadenylation signal fails to be used and/or RNA polymerase II fails to terminate. As a proof-of-principle, we show that RNAi-mediated depletion of well-established transcription termination factors, including the RNA endonuclease Cpsf73, results in increased circular RNA output from these plasmids in Drosophila and human cells. This method is generalizable as a circular RNA can be easily encoded downstream of any genomic region of interest. Circular RNA biomarkers, therefore, have great promise for identifying novel cellular factors and conditions that impact transcription termination processes.

An Integrated Systems Biology Approach Identifies the Proteasome as A Critical Host Machinery for ZIKV and DENV Replication.

The Zika virus (ZIKV) and dengue virus (DENV) flaviviruses exhibit similar replicative processes but have distinct clinical outcomes. A systematic understanding of virus-host protein-protein interaction networks can reveal cellular pathways critical to viral replication and disease pathogenesis. Here we employed three independent systems biology approaches toward this goal. First, protein array analysis of direct interactions between individual ZIKV/DENV viral proteins and 20,240 human proteins revealed multiple conserved cellular pathways and protein complexes, including proteasome complexes. Second, an RNAi screen of 10,415 druggable genes identified the host proteins required for ZIKV infection and uncovered that proteasome proteins were crucial in this process. Third, high-throughput screening of 6016 bioactive compounds for ZIKV inhibition yielded 134 effective compounds, including six proteasome inhibitors that suppress both ZIKV and DENV replication. Integrative analyses of these orthogonal datasets pinpoint proteasomes as critical host machinery for ZIKV/DENV replication. Our study provides multi-omics datasets for further studies of flavivirus-host interactions, disease pathogenesis, and new drug targets.

Silencing of ZFP36L2 increases sensitivity to temozolomide through G2/M cell cycle arrest and BAX mediated apoptosis in GBM cells.

Despite the advancements in primary brain tumour diagnoses and treatments, the mortality rate remains high, particularly in glioblastoma (GBM). Chemoresistance, predominantly in recurrent cases, results in decreased mean survival of patients with GBM. We aimed to determine the chemosensitisation and oncogenic characteristics of zinc finger protein 36-like 2 (ZFP36L2) in LN18 GBM cells via RNA interference (RNAi) delivery. We conducted a meta-analysis of microarray datasets and RNAi screening using pooled small interference RNA (siRNA) to identify the druggable genes responsive to GBM chemosensitivity. Temozolomide-resistant LN18 cells were used to evaluate the effects of gene silencing on chemosensitisation to the sub-lethal dose (1/10 of the median inhibitory concentration [IC50]) of temozolomide. ZFP36L2 protein expression was detected by western blotting. Cell viability, proliferation, cell cycle and apoptosis assays were carried out using commercial kits. A human apoptosis array kit was used to determine the apoptosis pathway underlying chemosensitisation by siRNA against ZFP36L2 (siZFP36L2). Statistical analyses were performed using one-way analysis of variance; p > 0.05 was considered significant. The meta-analysis and RNAi screening identified ZFP36L2 as a potential marker of GBM. ZFP36L2 knockdown significantly induced apoptosis (p < 0.05). Moreover, ZFP36L2 inhibition led to increased cell cycle arrest and decreased cell proliferation. Downstream analysis showed that the sub-lethal dose of temozolomide and siZFP26L2 caused major upregulation of BCL2-associated X, apoptosis regulator (BAX). ZFP36L2 has oncogenic and chemosensitive characteristics and may play an important role in gliomagenesis through cell proliferation, cell cycle arrest and apoptosis. This suggests that RNAi combined with chemotherapy treatment such as temozolomide may be a potential GBM therapeutic intervention in the future.

Overexpression of FKH-2/FOXG1 is neuroprotective in a C. elegans model of Machado-Joseph disease.

Machado-Joseph disease (MJD), also known as spinocerebellar ataxia type 3 (SCA3), is the most common form of dominantly inherited ataxia worldwide. This disease is caused by an expanded CAG repeat in the coding region of ATXN3. Due to our incomplete understanding of mechanisms and molecular pathways related to this disease, there are no therapies that successfully treat core MJD patients. Therefore, the identification of new candidate targets related to this disease is needed. In this study, we performed a large-scale RNA interference (RNAi) screen of 387 transcription factor genes leading to the identification of several modifiers (suppressors and enhancers) of impaired motility phenotypes in a mutant ATXN3 transgenic C. elegans model. We showed that inactivation of one particular gene, fkh-2/FOXG1, enhanced the motility defect, neurodegeneration and reduced longevity in our MJD models. Opposite to genetic inactivation, the overexpression of fkh-2 rescued the impaired motility, shortened-lifespan, and neurodegeneration phenotypes of mutant ATXN3 transgenics. We found that overexpression of FKH-2/FOXG1 in ATXN3 mutant worms is neuroprotective. Using our transgenic ATXN3 C. elegans models and the screening of an RNAi library, we gained insights into the pathways contributing to neurodegeneration, and found that FKH-2/FOXG1 has neuroprotective activity. These findings may aid the development of novel therapeutic interventions for MJD.

Enhanced Protein Damage Clearance Induces Broad Drug Resistance in Multitype of Cancers Revealed by an Evolution Drug-Resistant Model and Genome-Wide siRNA Screening.

Resistance to therapeutic drugs occurs in virtually all types of cancers, and the tolerance to one drug frequently becomes broad therapy resistance; however, the underlying mechanism remains elusive. Combining a whole whole-genome-wide RNA interference screening and an evolutionary drug pressure model with MDA-MB-231 cells, it is found that enhanced protein damage clearance and reduced mitochondrial respiratory activity are responsible for cisplatin resistance. Screening drug-resistant cancer cells and human patient-derived organoids for breast and colon cancers with many anticancer drugs indicates that activation of mitochondrion protein import surveillance system enhances proteasome activity and minimizes caspase activation, leading to broad drug resistance that can be overcome by co-treatment with a proteasome inhibitor, bortezomib. It is further demonstrated that cisplatin and bortezomib encapsulated into nanoparticle further enhance their therapeutic efficacy and alleviate side effects induced by drug combination treatment. These data demonstrate a feasibility for eliminating broad drug resistance by targeting its common mechanism to achieve effective therapy for multiple cancers.

RNAi Screening Identifies that TEX10 Promotes the Proliferation of Colorectal Cancer Cells by Increasing NF-κB Activation.

Colorectal cancer (CRC) has become a predominant cancer worldwide. To understand the process of carcinogenesis, a short hairpin RNA library screening is employed to search for candidate genes that promote proliferation in the CRC cell line HT29. The candidate genes overlap with differentially expressed genes in 32 CRC tumor tissues in the GEO dataset GSE8671. The seventh-ranked testis expressed 10 (TEX10) is upregulated in CRC and its knockdown decreases cell proliferation. The TEX10 high-expression group exhibits worse overall survival (P = 0.003) and progression-free survival (P = 0.001) than the TEX10 low-expression group. TEX10 depletion decreases the growth of CRC cells in vitro and in vivo. Gene set enrichment analysis indicates that the nuclear factor-kappa B pathway is significantly enriched in the genes downregulated by TEX10 knockdown. Mechanistically, TEX10 interacts with RELA and increases its nuclear localization. TEX10 promotes RELA occupancy at gene promoters and regulates the expression of a subset of RELA-targeted genes, including TNFAIP8, SAT1, and IL6ST. Taken together, this study identifies that TEX10 promotes the proliferation of CRC cells in an RELA-dependent manner. In addition, high TEX10 expression is associated with poor prognosis in CRC patients.

RNA Interference Screening Identifies Novel Roles for RhoBTB1 and RhoBTB3 in Membrane Trafficking Events in Mammalian Cells.

In the endomembrane system of mammalian cells, membrane traffic processes require a high degree of regulation in order to ensure their specificity. The range of molecules that participate in trafficking events is truly vast, and much attention to date has been given to the Rab family of small GTPases. However, in recent years, a role in membrane traffic for members of the Rho GTPase family, in particular Cdc42, has emerged. This prompted us to develop and apply an image-based high-content screen, initially focussing on the Golgi complex, using RNA interference to systematically perturb each of the 21 Rho family members and assess their importance to the overall organisation of this organelle. Analysis of our data revealed previously unreported roles for two atypical Rho family members, RhoBTB1 and RhoBTB3, in membrane traffic events. We find that depletion of RhoBTB3 affects the morphology of the Golgi complex and causes changes in the trafficking speeds of carriers operating at the interface of the Golgi and endoplasmic reticulum. In addition, RhoBTB3 was found to be present on these carriers. Depletion of RhoBTB1 was also found to cause a disturbance to the Golgi architecture, however, this phenotype seems to be linked to endocytosis and retrograde traffic pathways. RhoBTB1 was found to be associated with early endosomal intermediates, and changes in the levels of RhoBTB1 not only caused profound changes to the organisation and distribution of endosomes and lysosomes, but also resulted in defects in the delivery of two different classes of cargo molecules to downstream compartments. Together, our data reveal new roles for these atypical Rho family members in the endomembrane system.

Corrigendum: Chaperones, Membrane Trafficking and Signal Transduction Proteins Regulate Zaire Ebola Virus trVLPs and Interact With trVLP Elements.

[This corrects the article DOI: 10.3389/fmicb.2018.02724.].

Chaperones, Membrane Trafficking and Signal Transduction Proteins Regulate Zaire Ebola Virus trVLPs and Interact With trVLP Elements.

Ebolavirus (EBOV) life cycle involves interactions with numerous host factors, but it remains poorly understood, as does pathogenesis. Herein, we synthesized 65 siRNAs targeting host genes mostly connected with aspects of the negative-sense RNA virus life cycle (including viral entry, uncoating, fusion, replication, assembly, and budding). We produced EBOV transcription- and replication-competent virus-like particles (trVLPs) to mimic the EBOV life cycle. After screening host factors associated with the trVLP life cycle, we assessed interactions of host proteins with trVLP glycoprotein (GP), VP40, and RNA by co-immunoprecipitation (Co-IP) and chromatin immunoprecipitation (ChIP). The results demonstrate that RNAi silencing with 11 siRNAs (ANXA5, ARFGAP1, FLT4, GRP78, HSPA1A, HSP90AB1, HSPA8, MAPK11, MEK2, NTRK1, and YWHAZ) decreased the replication efficiency of trVLPs. Co-IP revealed nine candidate host proteins (FLT4, GRP78, HSPA1A, HSP90AB1, HSPA8, MAPK11, MEK2, NTRK1, and YWHAZ) potentially interacting with trVLP GP, and four (ANXA5, GRP78, HSPA1A, and HSP90AB1) potentially interacting with trVLP VP40. Ch-IP identified nine candidate host proteins (ANXA5, ARFGAP1, FLT4, GRP78, HSPA1A, HSP90AB1, MAPK11, MEK2, and NTRK1) interacting with trVLP RNA. This study was based on trVLP and could not replace live ebolavirus entirely; in particular, the interaction between trVLP RNA and host proteins cannot be assumed to be identical in live virus. However, the results provide valuable information for further studies and deepen our understanding of essential host factors involved in the EBOV life cycle.