Capturing Sialyl-glycan on Live Cancer Cells by Tailored Boronopeptide.

Boronic acid-containing molecules are substantially popularized in chemical biology and medicinal chemistry due to the broad spectrum of covalent conjugations as well as interaction modules offered by the versatile boron atom. Apparently, the WGA peptide (wheat germ agglutinin, 62-73), which shows a considerably low binding affinity to sialic acid, turned into a selective and >5 folds potent binder with the aid of a suitable boronic acid probe installed chemoselectively. In silico studies prompted us to install BA probes on the cysteine residue, supposedly located in close proximity to the bound sialic acid. In vitro studies revealed that the tailored boronopeptides show enhanced binding ability due to the synergistic recognition governed by selective non-covalent interactions and cis-diol boronic acid conjugation. The intense binding is observed even in 10 % serum, thus enabling profiling of sialyl-glycan on cancer cells, as compared with the widely used lectin, Sambucus nigra. The synergistic binding mode between the best boronopeptide (P3) binder and sialic acid was analyzed via 1 H and 11 B NMR.

Apoptosis-targeted gene therapy for non-small cell lung cancer using chitosan-poly-lactic-co-glycolic acid -based nano-delivery system and CASP8 and miRs 29A-B1 and 34A.

Non-small cell lung cancer (NSCLC) is a leading cause of cancer-related deaths worldwide, with resistance to apoptosis being a major driver of therapeutic resistance and aggressive phenotype. This study aimed to develop a novel gene therapy approach for NSCLC by targeting resistance to apoptosis. Loss of function mutations of caspase 8 (CASP8) and downregulation of microRNAs (miRs) 29A-B1 and 34A were identified as key contributors to resistance to apoptosis in NSCLC. A biodegradable polymeric nano-gene delivery system composed of chitosan-poly-lactic-co-glycolic acid was formulated to deliver initiator CASP8 and miRs 29A-B1 and 34A. The nano-formulation efficiently encapsulated the therapeutic genes effectively internalized into NSCLC cells and induced significant apoptosis. Evaluation of the nano-formulation in A549 tumor spheroids showed a significant increase in apoptosis within the core of the spheroids, suggesting effective penetration into the spheroid structures. We provide a novel nano-formulation that demonstrate therapeutic potential for suicidal gene therapy in NSCLC.

MiR-330-5p and miR-1270 target essential components of RNA polymerase I transcription and exhibit a novel tumor suppressor role in lung adenocarcinoma.

Upregulation of RNA polymerase I (Pol I) transcription and the overexpression of Pol I transcriptional machinery are crucial molecular alterations favoring malignant transformation. However, the causal molecular mechanism(s) of this aberration remain largely unknown. Here, we found that Pol I transcription and its core machinery are upregulated in lung adenocarcinoma (LUAD). We show that the loss of miRNAs (miR)-330-5p and miR-1270 expression contributes to the upregulation of Pol I transcription in LUAD. Constitutive overexpression of these miRs in LUAD cell lines suppressed the expression of core components of Pol I transcription, and reduced global ribosomal RNA synthesis. Importantly, miR-330-5p/miR-1270-mediated repression of Pol I transcription exerted multiple tumor suppressive functions including reduced proliferation, cell cycle arrest, enhanced apoptosis, reduced migration, increased drug sensitivity, and reduced tumor burden in a mouse xenograft model. Mechanistically, the downregulation of miR-330-5p and miR-1270 is regulated by Pol I subunit-derived circular RNA circ_0055467 and DNA hypermethylation, respectively. This study uncovers a novel miR-330-5p/miR-1270 mediated post-transcriptional regulation of Pol I transcription, and establish tumor suppressor properties of these miRs in LUAD. Ultimately, our findings provide a rationale for the therapeutic targeting of Pol I transcriptional machinery for LUAD.

Artificial intelligence uncovers carcinogenic human metabolites.

The genome of a eukaryotic cell is often vulnerable to both intrinsic and extrinsic threats owing to its constant exposure to a myriad of heterogeneous compounds. Despite the availability of innate DNA damage responses, some genomic lesions trigger malignant transformation of cells. Accurate prediction of carcinogens is an ever-challenging task owing to the limited information about bona fide (non-)carcinogens. We developed Metabokiller, an ensemble classifier that accurately recognizes carcinogens by quantitatively assessing their electrophilicity, their potential to induce proliferation, oxidative stress, genomic instability, epigenome alterations, and anti-apoptotic response. Concomitant with the carcinogenicity prediction, Metabokiller is fully interpretable and outperforms existing best-practice methods for carcinogenicity prediction. Metabokiller unraveled potential carcinogenic human metabolites. To cross-validate Metabokiller predictions, we performed multiple functional assays using Saccharomyces cerevisiae and human cells with two Metabokiller-flagged human metabolites, namely 4-nitrocatechol and 3,4-dihydroxyphenylacetic acid, and observed high synergy between Metabokiller predictions and experimental validations.

Salivary protein kinase C alpha and novel microRNAs as diagnostic and therapeutic resistance markers for oral squamous cell carcinoma in Indian cohorts.

Oral squamous cell carcinoma (OSCC) is the second leading cause of cancer-related morbidity and mortality in India. Tobacco, alcohol, poor oral hygiene, and socio-economic factors remain causative for this high prevalence. Identification of non-invasive diagnostic markers tailored for Indian population can facilitate mass screening to reduce overall disease burden. Saliva offers non-invasive sampling and hosts a plethora of markers for OSCC diagnosis. Here, to capture the OSCC-specific salivary RNA markers suitable for Indian population, we performed RNA-sequencing of saliva from OSCC patients (n = 9) and normal controls (n = 5). Differential gene expression analysis detected an array of salivary RNAs including mRNAs, long non-coding RNAs, transfer-RNAs, and microRNAs specific to OSCC. Computational analysis and functional predictions identified protein kinase c alpha (PRKCA), miR-6087, miR-449b-5p, miR-3656, miR-326, miR-146b-5p, and miR-497-5p as potential salivary indicators of OSCC. Notably, higher expression of PRKCA, miR-6087 and miR-449b-5p were found to be associated with therapeutic resistance and poor survival, indicating their prognostic potential. In addition, sequencing reads that did not map to the human genome, showed alignments with microbial reference genomes. Metagenomic and statistical analysis of these microbial reads revealed a remarkable microbial dysbiosis between OSCC patients and normal controls. Moreover, the differentially abundant microbial taxa showed a significant association with tumor promoting pathways including inflammation and oxidative stress. Summarily, we provide an integrated landscape of OSCC-specific salivary RNAs relevant to Indian population which can be instrumental in devising non-invasive diagnostics for OSCC.

EcTracker: Tracking and elucidating ectopic expression leveraging large-scale scRNA-seq studies.

Dramatic genomic alterations, either inducible or in a pathological state, dismantle the core regulatory networks, leading to the activation of normally silent genes. Despite possessing immense therapeutic potential, accurate detection of these transcripts is an ever-challenging task, as it requires prior knowledge of the physiological gene expression levels. Here, we introduce EcTracker, an R-/Shiny-based single-cell data analysis web server that bestows a plethora of functionalities that collectively enable the quantitative and qualitative assessments of bona fide cell types or tissue-specific transcripts and, conversely, the ectopically expressed genes in the single-cell ribonucleic acid sequencing datasets. Moreover, it also allows regulon analysis to identify the key transcriptional factors regulating the user-selected gene signatures. To demonstrate the EcTracker functionality, we reanalyzed the CRISPR interference (CRISPRi) dataset of the human embryonic stem cells differentiated into endoderm lineage and identified the prominent enrichment of a specific gene signature in the SMAD2 knockout cells whose identity was ambiguous in the original study. The key distinguishing features of EcTracker lie within its processing speed, availability of multiple add-on modules, interactive graphical user interface and comprehensiveness. In summary, EcTracker provides an easy-to-perform, integrative and end-to-end single-cell data analysis platform that allows decoding of cellular identities, identification of ectopically expressed genes and their regulatory networks, and therefore, collectively imparts a novel dimension for analyzing single-cell datasets.

Analysis of single-cell transcriptomes links enrichment of olfactory receptors with cancer cell differentiation status and prognosis.

Ectopically expressed olfactory receptors (ORs) have been linked with multiple clinically-relevant physiological processes. Previously used tissue-level expression estimation largely shadowed the potential role of ORs due to their overall low expression levels. Even after the introduction of the single-cell transcriptomics, a comprehensive delineation of expression dynamics of ORs in tumors remained unexplored. Our targeted investigation into single malignant cells revealed a complex landscape of combinatorial OR expression events. We observed differentiation-dependent decline in expressed OR counts per cell as well as their expression intensities in malignant cells. Further, we constructed expression signatures based on a large spectrum of ORs and tracked their enrichment in bulk expression profiles of tumor samples from The Cancer Genome Atlas (TCGA). TCGA tumor samples stratified based on OR-centric signatures exhibited divergent survival probabilities. In summary, our comprehensive analysis positions ORs at the cross-road of tumor cell differentiation status and cancer prognosis.

PDGFR-modulated miR-23b cluster and miR-125a-5p suppress lung tumorigenesis by targeting multiple components of KRAS and NF-kB pathways.

In NSCLC alterations in PDGF receptors are markers of worst prognosis and efficient targeting of these receptors is yet to be achieved. In this study, we explored PDGFR-regulated microRNAs demonstrating that miR-23b cluster and miR-125a-5p are downregulated by increased expression of PDGFR-α or PDGFR-ß in NSCLC cells. Mechanistically, the expression of these microRNAs is positively regulated by p53 and negatively modulated by NF-kB p65. Forced expression of miR-23b cluster or miR-125a-5p enhanced drug sensitivity and suppressed invasiveness of NSCLC cells by silencing several genes involved in oncogenic KRAS and NF-kB pathways, including SOS1, GRB2, IQGAP1, RALA, RAF-1, IKKß, AKT2, ERK2 and KRAS itself. Of note, an inverse correlation between miR-23b cluster, miR-125a-5p and respective target genes was also found in vivo in a large dataset of lung adenocarcinoma samples. Furthermore, in vivo delivery of miR-23b cluster or miR-125a-5p significantly repressed tumour growth in a highly aggressive NSCLC circulating tumour cell (CTC) patient derived explant (CDX) mouse model. In conclusion, our finding sheds light on the PDGFR signaling and endorses the possibility to employ miR-23b cluster and miR-125a-5p as therapeutic tools to silence simultaneously a range of redundant pathways and main effectors of tumorigenesis in NSCLC.

microRNAs: An Emerging Paradigm in Lung Cancer Chemoresistance.

Lung cancer is considered the most deadly of all cancers, with limited therapeutic options. Although advanced drugs have been tried in clinic, the therapeutic success has largely been hampered due to rapid development of drug-resistance mechanisms. Recently, microRNAs (miRNAs), a class of small non-coding RNAs, have occupied center stage in cancer biology. miRNAs negatively regulate gene expression either by promoting degradation or by interfering with translation of messenger RNA targets. Several lines of evidence have confirmed the crucial role of miRNAs in carcinogenesis, and, importantly, in the acquisition of resistance to chemotherapeutics. Modulation of miRNA expression levels has been proven to increase the efficacy of genotoxic drugs in various preclinical cancer studies. Therefore, comprehensive understanding of the role(s) of these key players in drug resistance may provide novel opportunities to design effective combinatorial therapeutic strategies for cancer treatment. In this review, we highlight recent findings on miRNAs acting as oncomiRs and tumor suppressor genes in lung cancer. Moreover, we discuss the involvement of miRNAs in different mechanisms of drug resistance in this deadly disease.

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

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

MiRNA-based therapeutic intervention of cancer.

MicroRNAs (miRNAs) are important modulators of eukaryotic gene expression. By targeting protein coding transcripts, miRNAs influence the cellular transcriptome and proteome, thus helping to determine cell fate. MiRNAs have emerged as crucial molecules in cancer research, in which recent studies have linked erratic expression of miRNAs to carcinogenesis and have provided solid evidence for their potential in cancer therapy. This review briefly summarises the recent knowledge on the involvement of miRNAs in tumourigenesis and reviews current studies on the therapeutic strategies and advances in the delivery of miRNAs.

TAF1B is a TFIIB-like component of the basal transcription machinery for RNA polymerase I.

Transcription by eukaryotic RNA polymerases (Pols) II and III and archaeal Pol requires structurally related general transcription factors TFIIB, Brf1, and TFB, respectively, which are essential for polymerase recruitment and initiation events. A TFIIB-like protein was not evident in the Pol I basal transcription machinery. We report that TAF1B, a subunit of human Pol I basal transcription factor SL1, is structurally related to TFIIB/TFIIB-like proteins, through predicted amino-terminal zinc ribbon and cyclin-like fold domains. SL1, essential for Pol I recruitment to the ribosomal RNA gene promoter, also has an essential postpolymerase recruitment role, operating through TAF1B. Therefore, a TFIIB-related protein is implicated in preinitiation complex assembly and postpolymerase recruitment events in Pol I transcription, underscoring the parallels between eukaryotic Pol I, II, and III and archaeal transcription machineries.

Bruton's tyrosine kinase is required for TLR-dependent heme oxygenase-1 gene activation via Nrf2 in macrophages.

Heme oxygenase (HO)-1 is the inducible isoform of the rate-limiting enzyme of heme degradation and provides cytoprotection against oxidative stress by its products carbon monoxide and biliverdin. More recently, HO-1 has also been shown to exert immunomodulatory functions via cell type-specific anti-inflammatory effects in myeloid/macrophage cells. In the current study, it is demonstrated that Bruton's tyrosine kinase (Btk), the gene of which is mutated in the human immunodeficiency X-linked agammaglobulinemia, is involved in the upregulation of HO-1 gene expression via TLR signaling in macrophages. The specific Btk inhibitor LFM-A13 blocked HO-1 induction by the classical TLR4 ligand LPS in cell cultures of RAW264.7 monocytic cells and primary mouse alveolar macrophages. Moreover, upregulation of HO-1 gene expression was abrogated in LPS-stimulated alveolar macrophages from Btk(-/-) mice. Transfection studies with luciferase reporter gene constructs demonstrated that LPS-dependent induction of HO-1 promoter activity was attenuated by pharmacological Btk inhibition and by an overexpressed dominant-negative mutant of Btk. This induction was mediated by the transcription factor Nrf2, which is a master regulator of the antioxidant cellular defense. Accordingly, nuclear translocation of Nrf2 in LPS-treated macrophages was reduced by Btk inhibition. The generation of reactive oxygen species, but not that of NO, was involved in this regulatory pathway. Btk-dependent induction of HO-1 gene expression was also observed upon macrophage stimulation with ligands of TLR2, TLR6, TLR7, and TLR9, suggesting that Btk is required for HO-1 gene activation by major TLR pathways.

Inhibition and genetic deficiency of p38 MAPK up-regulates heme oxygenase-1 gene expression via Nrf2.

Heme oxygenase (HO)-1 is the inducible isoform of the first and rate-limiting enzyme of heme degradation. The HO products carbon monoxide and bilirubin not only provide antioxidant cytoprotection, but also have potent anti-inflammatory and immunomodulatory functions. Although HO-1 has previously been shown to be induced by various stimuli via activation of the p38 MAPK signaling pathway, the role of this protein kinase for HO-1 gene regulation is largely unknown. In the present study, it is demonstrated that pharmacological inhibitors of p38 induced HO-1 expression in monocytic cells. Moreover, basal HO-1 gene expression levels were markedly higher in untreated murine embryonic fibroblasts (MEF) from p38alpha(-/-) mice compared with those from wild-type mice. Transfection studies with luciferase reporter gene constructs indicate that increased HO-1 gene expression via inhibition of p38 was mediated by the transcription factor Nrf2, which is a central regulator of the cellular oxidative stress response. Accordingly, inhibitors of p38 induced binding of nuclear proteins to a Nrf2 target sequence of the HO-1 promoter, but did not affect HO-1 protein expression and promoter activity in Nrf2(-/-) MEF. Genetic deficiency of p38 led to enhanced phosphorylation of ERK and increased cellular accumulation of reactive oxygen species. In addition, pharmacological blockage of ERK and scavenging of reactive oxygen species with N-acetylcysteine reduced HO-1 gene expression in p38(-/-) MEF, respectively. Taken together, it is demonstrated that pharmacological inhibition and genetic deficiency of p38 induce HO-1 gene expression via a Nrf2-dependent mechanism in monocytic cells and MEF.

Transcriptional induction of junctional adhesion molecule-C gene expression in activated T cells.

Junctional adhesion molecule (JAM)-C is an Ig superfamily protein, which is involved in the regulation of various inflammatory and vascular events such as transendothelial leukocyte migration. JAM-C is expressed highly on the surface of endothelial cells and platelets, whereas expression in T lymphocytes is not well studied. To investigate the specific gene regulation of JAM-C in T lymphocytes, we determined JAM-C expression in quiescent and activated human T cells. Treatment with the polyclonal T cell activator PHA increased surface and total JAM-C expression in T cells time- and dose-dependently, as determined by flow cytometry and immunoblot analysis. In contrast, no up-regulation of JAM-A in activated T cells was detectable. The highest level of JAM-C up-regulation by PHA was observed in CD3(+)forkhead box P3(+) and CD4(+)CD25(high) T cells. Moreover, TCR activation with combined anti-CD3 and anti-CD28 stimulation induced JAM-C expression in T cells. JAM-C induction occurred at the mRNA level, suggesting a transcriptional regulatory mechanism of JAM-C expression. Accordingly, we studied the regulation of the human JAM-C gene promoter in transiently transfected T cells. Luciferase activity of a JAM-C promoter gene construct with three potential consensus sites for the transcription factor NFAT was induced markedly in activated T cells. Finally, pretreatment with two pharmacological inhibitors of calcineurin, cyclosporin A, and FK-506, but not with MAPK inhibitors, blocked JAM-C induction in activated T cells. In summary, JAM-C is up-regulated in activated human T lymphocytes via a transcriptional mechanism, suggesting a potential role of JAM-C in T cell functions.

An atypical NF-kappa B-regulated pathway mediates phorbol ester-dependent heme oxygenase-1 gene activation in monocytes.

Heme oxygenase (HO)-1 catalyzes the rate-limiting step of heme degradation and plays an important anti-inflammatory role via its enzymatic products carbon monoxide and biliverdin. In this study it is reported that the HO-1 gene is transcriptionally induced by the phorbol ester PMA in cell cultures of monocytic cells with a regulatory pattern that is different from that of LPS-dependent HO-1 induction in these cells. Activation of HO-1 by PMA was mediated via a newly identified kappaB element of the proximal rat HO-1 gene promoter region (-284 to -275). This HO-kappaB element was a nuclear target for the NF-kappaB subunit p65/RelA as determined by nuclear binding assays and transfection experiments with luciferase reporter gene constructs in RAW264.7 monocytes. Moreover, PMA-dependent induction of endogenous HO-1 gene expression and promoter activity was abrogated in embryonic fibroblasts from p65(-/-) mice. PMA-dependent HO-1 gene activation was reduced by an overexpressed dominant negative mutant of IkappaBalpha, but not by dominant negative IkappaB kinase-2, suggesting that the classical NF-kappaB pathway was not involved in this regulation. The antioxidant N-acetylcysteine and inhibitors of p38 MAPK or serine/threonine kinase CK2 blocked PMA-dependent HO-1 gene activation. Finally, it is demonstrated by luciferase assays with a Gal4-CHOP fusion protein that the activation of p38 MAPK by PMA was independent of CK2. Taken together, induction of HO-1 gene expression by PMA is regulated via an IkappaB kinase-independent, atypical NF-kappaB pathway that is mediated via the activation of p38 MAPK and CK2.

Inhibition of phorbol ester-dependent peroxiredoxin I gene activation by lipopolysaccharide via phosphorylation of RelA/p65 at serine 276 in monocytes.

Peroxiredoxin I (Prx I) is an antioxidant enzyme with thioredoxin-dependent peroxidase activity which is involved in various cellular processes such as regulation of cell proliferation. Here, it is shown that the proinflammatory mediator lipopolysaccharide (LPS) inhibits the induction of Prx I expression and promoter activity by the phorbol ester 12-O-tetradecanoylphorbol- 13-acetate (TPA) in RAW264.7 monocytes, but not that of cyclooxygenase-2. LPS-dependent repression of Prx I induction by TPA was mediated via a newly identified kappaB site in the Prx I promoter, but the "classical" NF-kappaB cascade was not involved in this regulatory pathway, because IkappaB did not affect LPS-mediated Prx I repression. By contrast, phosphorylation of p65 at serine 276, which enhances the transcriptional activity of NF-kappaB, was up-regulated by TPA and was reduced by simultaneous exposure to LPS. Functional studies with Gal4-p65 constructs revealed that serine 276 is crucial to confer LPS-dependent repression of TPA-mediated induction of p65 transactivation. Finally, repression of TPA-dependent Prx I induction by LPS was mediated via Bruton's tyrosine kinase as indicated by studies with the pharmacological inhibitor LFM-A13. In summary, LPS-dependent inhibition of Prx I gene activation by TPA in monocytes is regulated via a pathway that involves phosphorylation of the NF-kappaB subunit p65 at serine 276.