AMPK activation reduces cancer cell aggressiveness via inhibition of monoamine oxidase A (MAO-A) expression/activity.

AIM: AMPK can be considered as an important target molecule for cancer for its unique ability to directly recognize cellular energy status. The main aim of this study is to explore the role of different AMPK activators in managing cancer cell aggressiveness and to understand the mechanistic details behind the process. MAIN METHODS: First, we explored the AMPK expression pattern and its significance in different subtypes of lung cancer by accessing the TCGA data sets for LUNG, LUAD and LUSC patients and then established the correlation between AMPK expression pattern and overall survival of lung cancer patients using Kaplan-Meire plot. We further carried out several cell-based assays by employing different wet lab techniques including RT-PCR, Western Blot, proliferation, migration and invasion assays to fulfil the aim of the study. KEY FINDINGS: SIGNIFICANCE: This study identifies the importance of AMPK activators as a repurposing agent for combating lung and colon cancer cell aggressiveness. It also suggests SRT-1720 as a potent repurposing agent for cancer treatment especially in NSCLC patients where a point mutation is present in LKB1.

Rac1 plays a crucial role in MCP-1-induced monocyte adhesion and migration.

Monocyte migration is an important process in inflammation and atherogenesis. Identification of the key signalling pathways that regulate monocyte migration can provide prospective targets for prophylactic treatments in inflammatory diseases. Previous research showed that the focal adhesion kinase Pyk2, Src kinase and MAP kinases play an important role in MCP-1-induced monocyte migration. In this study, we demonstrate that MCP-1 induces iPLA2 activity, which is regulated by PKCß and affects downstream activation of Rac1 and Pyk2. Rac1 interacts directly with iPLA2 and Pyk2, and plays a crucial role in MCP-1-mediated monocyte migration by modulating downstream Pyk2 and p38 MAPK activation. Furthermore, Rac1 is necessary for cell spreading and F-actin polymerization during monocyte adhesion to fibronectin. Finally, we provide evidence that Rac1 controls the secretion of inflammatory mediator vimentin from MCP-1-stimulated monocytes. Altogether, this study demonstrates that the PKCß/iPLA2/Rac1/Pyk2/p38 MAPK signalling cascade is essential for MCP-1-induced monocyte adhesion and migration.

Regulation of RNA methylation by therapy treatment, promotes tumor survival.

Our data previously revealed that chemosurviving cancer cells translate specific genes. Here, we find that the m6A-RNA-methyltransferase, METTL3, increases transiently in chemotherapy-treated breast cancer and leukemic cells in vitro and in vivo. Consistently, m6A increases on RNA from chemo-treated cells, and is needed for chemosurvival. This is regulated by eIF2α phosphorylation and mTOR inhibition upon therapy treatment. METTL3 mRNA purification reveals that eIF3 promotes METTL3 translation that is reduced by mutating a 5'UTR m6A-motif or depleting METTL3. METTL3 increase is transient after therapy treatment, as metabolic enzymes that control methylation and thus m6A levels on METTL3 RNA, are altered over time after therapy. Increased METTL3 reduces proliferation and anti-viral immune response genes, and enhances invasion genes, which promote tumor survival. Consistently, overriding phospho-eIF2α prevents METTL3 elevation, and reduces chemosurvival and immune-cell migration. These data reveal that therapy-induced stress signals transiently upregulate METTL3 translation, to alter gene expression for tumor survival.

Ribosome changes reprogram translation for chemosurvival in G0 leukemic cells.

Quiescent leukemic cells survive chemotherapy, with translation changes. Our data reveal that FXR1, a protein amplified in several aggressive cancers, is elevated in quiescent and chemo-treated leukemic cells and promotes chemosurvival. This suggests undiscovered roles for this RNA- and ribosome-associated protein in chemosurvival. We find that FXR1 depletion reduces translation, with altered rRNAs, snoRNAs, and ribosomal proteins (RPs). FXR1 regulates factors that promote transcription and processing of ribosomal genes and snoRNAs. Ribosome changes in FXR1-overexpressing cells, including RPLP0/uL10 levels, activate eIF2α kinases. Accordingly, phospho-eIF2α increases, enabling selective translation of survival and immune regulators in FXR1-overexpressing cells. Overriding these genes or phospho-eIF2α with inhibitors reduces chemosurvival. Thus, elevated FXR1 in quiescent or chemo-treated leukemic cells alters ribosomes that trigger stress signals to redirect translation for chemosurvival.

Long-read sequencing reveals complex patterns of wraparound transcription in polyomaviruses.

Polyomaviruses (PyV) are ubiquitous pathogens that can cause devastating human diseases. Due to the small size of their genomes, PyV utilize complex patterns of RNA splicing to maximize their coding capacity. Despite the importance of PyV to human disease, their transcriptome architecture is poorly characterized. Here, we compare short- and long-read RNA sequencing data from eight human and non-human PyV. We provide a detailed transcriptome atlas for BK polyomavirus (BKPyV), an important human pathogen, and the prototype PyV, simian virus 40 (SV40). We identify pervasive wraparound transcription in PyV, wherein transcription runs through the polyA site and circles the genome multiple times. Comparative analyses identify novel, conserved transcripts that increase PyV coding capacity. One of these conserved transcripts encodes superT, a T antigen containing two RB-binding LxCxE motifs. We find that superT-encoding transcripts are abundant in PyV-associated human cancers. Together, we show that comparative transcriptomic approaches can greatly expand known transcript and coding capacity in one of the simplest and most well-studied viral families.

Fatty acids and their lipid mediators in the induction of cellular apoptosis in cancer cells.

The oxygenation of polyunsaturated fatty acids such as arachidonic and linoleic acid through enzymes like lipoxygenases (LOXs) are common and often leads to the production of various bioactive lipids that are important both in acute inflammation and its resolution and thus in disease progression. Amongst the several isoforms of LOX that are expressed in mammals, 15-lipoxygenase (15-LOX) has shown to be crucial in the context of inflammation. Moreover, being expressed in cells of the immune system, as well as in epithelial cells; the enzyme has been shown to crosstalk with a number of important signalling pathways. Mounting evidences from recent reports suggest that 15-LOX has anti-cancer activities which are dependent or independent of its metabolites, and is executed through several downstream pathways like cGMP, PPAR, p53, p21 and NAG-1. However, it is still unclear whether the up-regulation of 15-LOX is associated with cancer cell apoptosis. Monoamine oxidase A (MAO-A), on the other hand, is a mitochondrial flavoenzyme which is believed to be involved in the pathogenesis of atherosclerosis and inflammation and in many other neurological disorders. MAO-A has also been reported as a potential therapeutic target in different types of cancers like prostate cancer, lung cancer etc. In this review, we discussed about the role of fatty acids and their lipid mediators in cancer cell apoptosis. Here we particularly focused on the contribution of oxidative enzymes like 15-LOX and MAO-A in mediating apoptosis in lung cancer cell after fatty acid induction.

Localized outbreaks of Pseudomonas aeruginosa belonging to international high-risk clones in a south Indian hospital.

Introduction. Pseudomonas aeruginosa is now considered as a major bacterial pathogen associated with hospital infections. Frequently, multidrug-resistant (MDR) and extensively drug-resistant (XDR) P. aeruginosa are being encountered. Unusual increase in the P. aeruginosa infections led to the suspicion of outbreaks in the urology ward and cardiothoracic and vascular surgery intensive care unit (CTVS-ICU).Hypothesis. We hypothesize that the localized outbreaks may have originated from environmental sources within the hospital premises. An alternative possibility is the transmission from a previously infected patient or hospital attendant. Understanding the drug-resistance profile and genome characteristics of these clinical samples would determine the likely source of infection and spread.Aim. To perform epidemiological and molecular investigations on the suspected outbreaks of P. aeruginosa in the study centre and identify potential sources of infection.Methodology. Fourteen drug-resistant P. aeruginosa isolated from patients of the urology ward, CTVS-ICU and tap waters collected during the suspected outbreaks were subjected to microbiological and genomic analysis. Comparative genome (CG) analysis of these 14 study genomes with 284 complete P. aeruginosa genomes was performed.Results. Multilocus sequence typing analysis revealed that the isolates belonged to five different sequence types (ST235, ST357, ST639, ST654 and ST1203) and clustered into three distinct groups while two CTVS-ICU isolates remained as singletons. Genome analysis distinguished that the outbreaks in the urology ward and CTVS-ICU are independent, epidemiologically unrelated to each other and with the tap-water isolates.Conclusion. This study highlights the presence of distinct, clonally unrelated, drug-resistant P. aeruginosa within a hospital setting. The genome analysis of the two localized outbreaks revealed their distinct genetic background and phylogenetically unrelated origin. Vigilant screening and effective implementation of infection control measures led to the successful containment of potential environmental reservoirs of P. aeruginosa within the premises.

NapA (Rv0430), a Novel Nucleoid-Associated Protein that Regulates a Virulence Operon in Mycobacterium tuberculosis in a Supercoiling-Dependent Manner.

Comparison of Mycobacterium tuberculosis with Escherichia coli reveals a reduction in the diversity of DNA-managing proteins, such as DNA topoisomerases, although genome sizes are similar for the two species. The same is true for nucleoid-associated proteins (NAPs), important factors in bacterial chromosome compaction, chromosome remodeling, and regulation of gene expression. In a search for still uncharacterized NAPs, we found that M. tuberculosis protein Rv0430 has NAP-like features: it binds to DNA in a length- and supercoil-dependent fashion, prefers A/T-rich DNA sequences, protects DNA from damaging agents, and modulates DNA supercoiling. At a ratio of 1 dimer/40 bps of DNA, Rv0430 bridges distant DNA segments; at 1 dimer/20 bps, it coats DNA, forming inflexible rods. Rv0430 also stimulates the DNA relaxation activity of topoisomerase I. Remarkably, Rv0430 stimulates its own promoter in a supercoil-dependent manner. It is the first gene of an operon harboring two regulators of M. tuberculosis virulence (virR and sodC), and controls the expression of these downstream virulence regulators and therefore itself is a virulence regulator. The sensitivity of rv0430 expression to supercoiling is consistent with supercoiling being important for infection by M. tuberculosis. Thus, Rv0430 is a novel NAP, doubling up as a topology modulator of M. tuberculosis.

Physical and functional interaction between nucleoid-associated proteins HU and Lsr2 of Mycobacterium tuberculosis: altered DNA binding and gene regulation.

Nucleoid-associated proteins (NAPs) in bacteria contribute to key activities such as DNA compaction, chromosome organization and regulation of gene expression. HU and Lsr2 are two principal NAPs in Mycobacterium tuberculosis (Mtb). HU is essential for Mtb survival and is one of the most abundant NAPs. It differs from other eubacterial HU proteins in having a long, flexible lysine- and arginine-rich carboxy-terminal domain. Lsr2 of Mtb is the functional analogue of the bacterial NAP commonly called H-NS. Lsr2 binds to and regulates expression of A/T-rich portions of the otherwise G/C-rich mycobacterial chromosome. Here, we demonstrate that HU and Lsr2 interact to form a complex. The interaction occurs primarily through the flexible carboxy-terminal domain of HU and the acidic amino-terminal domain of Lsr2. The resulting complex, upon binding to DNA, forms thick nucleoprotein rods, in contrast to the DNA bridging seen with Lsr2 and the DNA compaction seen with HU. Furthermore, transcription assays indicate that the HU-Lsr2 complex is a regulator of gene expression. This physical and functional interaction between two NAPs, which has not been reported previously, is likely to be important for DNA organization and gene expression in Mtb and perhaps other bacterial species.

Genome-wide mRNA-miRNA profiling uncovers a role of the microRNA miR-29b-1-5p/PHLPP1 signalling pathway in Helicobacter pylori-driven matrix metalloproteinase production in gastric epithelial cells.

Aberrant expression of microRNAs (miRNAs) is associated with tumour progression, extracellular matrix remodelling, and cell proliferation. miRNAs modulate host gene expression during infection by pathogens such as Helicobacter pylori, which is associated with varying degrees of gastric pathology. In order to gain insight into the regulation of gene expression by miRNAs during H. pylori infection of gastric epithelial cells and its likely downstream consequences, we analysed the transcriptomes and miRnomes of AGS cells infected with H. pylori. In silico analysis of miRNA-mRNA interactions suggested that miR-29b-1-5p was a likely regulator of pathways associated with gastric epithelial cell pathology. We validated PH domain leucine rich phosphatase 1 (PHLPP1), a negative regulator of the Akt signalling pathway, as a target of miR-29b-1-5p. In an in vivo mouse model, we observed that infection with H. pylori was associated with upregulation of miR-29b-1-5p and downregulation of PHLPP1. Transfection with either a mimic or an inhibitor of miR-29b-1-5p confirmed that downregulation of PHLPP1 upregulates Akt-dependent NF-κB signalling leading to activation of matrix metalloproteinases 2 and 9, players in the degradation of extracellular matrix during H. pylori infection. The secreted antigen HP0175 was associated with upregulation of miR-29b-1-5p, regulation of metalloproteinase activity, and migration of AGS cells. Our study suggests that targeting the miR-29b-1-5p/PHLPP1 signalling axis could be a potential host-directed approach for regulating the outcome of H. pylori infection.

Diarylethenes Display In Vitro Anti-TB Activity and Are Efficient Hits Targeting the Mycobacterium tuberculosis HU Protein.

Tuberculosis continues to be a great source of concern in global health because of the large reservoir of humans infected with the bacilli and the appearance of clinical isolates resistant to a wide array of anti-tuberculosis drugs. New drugs with novel mechanisms of action on new targets are urgently required to reduce global tuberculosis burden. Mycobacterium tuberculosis nucleoid associated protein (NAP) HU has been shown to be druggable and essential for the organism's survival. In this study, four diarylethenes were synthesized using a one-pot decarboxylated Heck-coupling of coumaric acids with iodoanisoles. The prepared compounds 1-4 were tested for their in vitro growth inhibition of M. tuberculosis H37Rv using the spot culture growth inhibition assay, displaying minimum inhibitory concentrations between 9 and 22 µM. Their cytotoxicity against BHK-21 cell line showed half inhibition at concentrations between 98 and 729 µM. The most selective hit (SI = 81), demonstrated inhibition of M. tuberculosis HU protein involved in maintaining bacterial genome architecture.

The secreted antigen, HP0175, of Helicobacter pylori links the unfolded protein response (UPR) to autophagy in gastric epithelial cells.

Autophagy is an intracellular catabolic process that is required to maintain cellular homeostasis. Pathogen-elicited host cell autophagy may favour containment of infection or may help in bacterial survival. Pathogens have developed the ability to modulate host autophagy. The secreted antigen HP0175, a peptidyl prolyl cis,trans isomerase of Helicobacter pylori, has moonlighting functions with reference to host cells. Here we show that it executes autophagy in gastric epithelial cells. Autophagy is dependent on the unfolded protein response (UPR) that activates the expression of PKR-like ER kinase (PERK). This is accompanied by phosphorylation of eukaryotic initiation factor 2α (eIF-2α) and transcriptional activation of ATF4 and CHOP. Knockdown of UPR-related genes inhibits the conversion of LC3I to LC3II, a marker of autophagy. The autophagy-inducing ability of H. pylori is compromised when cells are infected with an isogenic hp0175 mutant. Autophagy precedes apoptosis. Silencing of BECLIN1 augments cleavage of caspase 3 as well as apoptosis. Increased apoptosis of gastric epithelial cells is known to be linked to H. pylori-mediated gastric inflammation and carcinogenesis. To the best of our knowledge, this study provides the first demonstration of how HP0175 endowed with moonlighting functions links UPR-dependent autophagy and apoptosis during H. pylori infection.