New oncogenic functions of LINE1 retroelement as a ceRNA for tumor suppressive microRNA miR-126 on ENPP5.

Retroelements (REs) had been considered 'Junk' until the encyclopedia of DNA elements (ENCODE) project demonstrated that most genome is functional. Although the function of retroelements has been reported in diverse cancers including human breast cancer (HBC) and subtypes, only a few studies have suggested the putative functions of REs via their random genome integration. A canine mammary tumor (CMT) has been highlighted due to the similarities in molecular and pathophysiology with HBC. This study investigated the putative roles of REs common in both HBC and CMT. The human LINE and HERV-K sequences harbor many miRNAs responsive elements (MREs) for tumor-suppressive miRNA such as let-7. We also observed that various MREs are exist in the ERV and LINE highly expressed in the transcriptome data of CMT as well as HBC sets. MREs against miR-126 were highly expressed in both HBC and CMT while the levels of miR-126 were down-regulated. Oppositely, the expression of miR-126 target genes was significantly up-regulated in the cancers. Moreover, cancer patients with an increased level of miR-126 showed better overall survival. The expression of ENPP5, a putative miR-126 target gene, was downregulated by miR-126 mimic. Importantly, overexpression of LINE fragment significantly suppressed miR-126 function on the target gene expression. We propose the functional role of REs expression in tumorigenesis as competing endogenous RNAs (ceRNA) against tumor-suppressive miRNAs. This study provided pieces of evidence that LINE expression, even partial and fragmented, have a regulatory function in ENPP5 gene expression via the competition with miR-126.

Compounds That Have an Anti-Biofilm Effect against Common Bacteria at Very Low Concentrations and Their Antibiotic Combination Effect.

Two synthetic compounds, MHY1383, azo-resveratrol and MHY1387, 5-[4-hydroxy-3,5-methoxybenzy]-2-thioxodihydropyrimidine-4,6[1H,5H]-dione have been reported to have an anti-biofilm effect on Pseudomonas aeruginosa at very low concentrations (1-10 pM). Here, we investigated the anti-biofilm effects of these compounds in various bacteria. We found that MHY1383 significantly inhibited Escherichia coli, Bacillus subtilis, and Staphylococcus aureus biofilm formation at 1 pM, 1 nM, and 10 nM, respectively. MHY1387 also inhibited the biofilm formation of E. coli, B. subtilis, and S. aureus at 1 pM, 10 nM, and 100 pM, respectively. Both MHY1383 and MHY1387 showed medium-dependent anti-biofilm effects on Salmonella enterica at high concentrations (10 µM). We also tested the susceptibility to antibiotics by measuring the minimum inhibitory concentration (MIC) in various bacteria. When P. aeruginosa, E. coli, B. subtilis, S. enterica, and S. aureus were treated with MHY1383 or MHY1387 in combination with four different antibiotics, the MICs of carbenicillin against B. subtilis and S. aureus were lowered more than two-fold by the combination with MHY1387. However, in all other combinations, the MIC changed within two-fold. The results of this study suggest that MHY1383 and MHY1387 are effective anti-biofilm agents and can be used at very low concentrations against biofilms formed by various types of bacteria. We also suggest that even if a substance that inhibits biofilm is used together with antibiotics, it does not necessarily have the effect of lowering the MIC of the antibiotics.

ABCA9, an ER cholesterol transporter, inhibits breast cancer cell proliferation via SREBP-2 signaling.

The association between cholesterol metabolism and cancer development and progression has been recently highlighted. However, the role and function of many cholesterol transporters remain largely unknown. Here, we focused on the ATP-binding cassette subfamily A member 9 (ABCA9) transporter given that its expression is significantly downregulated in both canine mammary tumors and human breast cancers, which in breast cancer patients correlates with poor prognosis. We found that ABCA9 is mainly present in the endoplasmic reticulum (ER) and is responsible for promoting cholesterol accumulation in this structure. Accordingly, ABCA9 inhibited sterol-regulatory element binding protein-2 (SREBP-2) translocation from the ER to the nucleus, a crucial step for cholesterol synthesis, resulting in the downregulation of cholesterol synthesis gene expression. ABCA9 expression in breast cancer cells attenuated cell proliferation and reduced their colony-forming abilities. We identified ABCA9 expression to be regulated by Forkhead box O1 (FOXO1). Inhibition of PI3K induced enhanced ABCA9 expression through the activation of the PI3K-Akt-FOXO1 pathway in breast cancer cells. Altogether, our study suggests that ABCA9 functions as an ER cholesterol transporter that suppresses cholesterol synthesis via the inhibition of SREBP-2 signaling and that its restoration halts breast cancer cell proliferation. Our findings provide novel insight into the vital role of ABCA9 in breast cancer progression.

Anthranilate Acts as a Signal to Modulate Biofilm Formation, Virulence, and Antibiotic Tolerance of Pseudomonas aeruginosa and Surrounding Bacteria.

Anthranilate is a diffusible molecule produced by Pseudomonas aeruginosa and accumulates as P. aeruginosa grows. Anthranilate is an important intermediate for the synthesis of tryptophan and the Pseudomonas quinolone signal (PQS), as well as metabolized by the anthranilate dioxygenase complex (antABC operon products). Here we demonstrate that anthranilate is a key factor that modulates the pathogenicity-related phenotypes of P. aeruginosa and other surrounding bacteria in the environment, such as biofilm formation, antibiotic tolerance, and virulence. We found that the anthranilate levels in P. aeruginosa cultures rapidly increased in the stationary phase and then decreased again, forming an anthranilate peak. Biofilm formation, antibiotic susceptibility, and virulence of P. aeruginosa were significantly altered before and after this anthranilate peak. In addition, these phenotypes were all modified by the mutation of antABC and exogenous addition of anthranilate. Anthranilate also increased the antibiotic susceptibility of other species of bacteria, such as Escherichia coli, Salmonella enterica, Bacillus subtilis, and Staphylococcus aureus. Before the anthranilate peak, the low intracellular anthranilate level was maintained through degradation from the antABC function, in which induction of antABC was also limited to a small extent. The premature degradation of anthranilate, due to its high levels, and antABC expression early in the growth phase, appears to be toxic to the cells. From these results, we propose that by generating an anthranilate peak as a signal, P. aeruginosa may induce some sort of physiological change in surrounding cells. IMPORTANCE Pseudomonas aeruginosa is a notorious pathogen with high antibiotic resistance, strong virulence, and ability to cause biofilm-mediated chronic infection. We found that these characteristics change profoundly before and after the time when anthranilate is produced as an "anthranilate peak". This peak acts as a signal that induces physiological changes in surrounding cells, decreasing their antibiotic tolerance and biofilm formation. This study is important in that it provides a new insight into how microbial signaling substances can induce changes in the pathogenicity-related phenotypes of cells in the environment. In addition, this study shows that anthranilate can be used as an adjuvant to antibiotics.

Antipathogenic Compounds That Are Effective at Very Low Concentrations and Have Both Antibiofilm and Antivirulence Effects against Pseudomonas aeruginosa.

Pseudomonas aeruginosa, a human pathogen, causes both acute and chronic infections that are mediated by virulence factor production and biofilm formation. Since both characteristics of P. aeruginosa are regulated by quorum sensing (QS), we screened 126 synthetic chemicals for anti-QS activity and finally selected the compounds that have both antivirulence and antibiofilm activities. To efficiently screen the chemical library, the following reporter-based bioassay systems were used: the QS- or biofilm-specific promoter-lacZ fusions (lasIp- or PA1897p-lacZ for the QS activity and cdrAp-lacZ for measuring the intracellular c-di-GMP levels). We also measured the production of virulence factors and biofilm formation in P. aeruginosa. A small-animal infection model using mealworms was also used for virulence analysis. From this screening, MHY1383 and MHY1387 were found to have both antivirulence and antibiofilm activities in P. aeruginosa. Most importantly, MHY1383 and MHY1387 exhibited these activities at very low concentrations, showing a significant anti-QS effect at 100 pM and an antibiofilm effect at 1 to 10 pM. By treating P. aeruginosa with these compounds, the virulence factor production and biofilm formation of P. aeruginosa were significantly reduced. These compounds can be developed as promising antipathogenic and antibiofilm drugs that can be applied in situations where such compounds must be used in an extremely low concentration. Our findings also offer a significant advantage for developing therapeutic agents with few adverse side effects. IMPORTANCE Many antibiotics are increasingly losing their efficacy due to antibiotic resistance mediated by biofilm formation. In this study, we screened a synthetic chemical library and discovered several compounds that have both antivirulence and antibiofilm effects against Pseudomonas aeruginosa, a notorious human pathogen. Two of them had these effects at extremely low concentrations and are expected not to develop resistance, unlike conventional antibiotics, because they have no effect on the growth of bacteria. Our results strongly suggest that these compounds act on the target in a noncompetitive manner, indicating that they are distinct from other previously known quorum sensing inhibitors or biofilm inhibitors. Our findings offer a significant advantage for developing therapeutic agents with few adverse side effects.

Long Non-Coding RNA Associated with Cholesterol Homeostasis and Its Involvement in Metabolic Diseases.

Cholesterol is an essential cell component that functions to create and maintain all kinds of cell membranes and lipoprotein particles. It is crucial to maintain the proper amount of cholesterol at both the cellular and systemic level. Recently, the importance of cholesterol has been reported not only in various cell development processes but also in the development of diseases. Furthermore, the involvement of long non-coding RNAs (lncRNAs), which are regarded as important epigenetic regulators in gene expression, has also been reported in cholesterol homeostasis. It is thus necessary to summarize the research on lncRNAs related to cholesterol with increased interest. This review organized the role of lncRNAs according to the major issues in cholesterol homeostasis: efflux, metabolism and synthesis, and disease process.

Thermoregulation of Pseudomonas aeruginosa Biofilm Formation.

We investigated the effect of temperature on the biofilm formation of Pseudomonas aeruginosa and revealed that the biofilm formation increased rapidly at temperatures lower than 25°C. P. aeruginosa formed the most robust biofilm of a conspicuous mushroom-like structure at 20°C. However, when the temperature increased to 25°C, the biofilm formation rapidly decreased. Above 25°C, as the temperature rose, the biofilm formation increased again little by little despite its less-structured form, indicating that 25°C is the low point of biofilm formation. The intracellular 3',5'-cyclic diguanylate (c-di-GMP) levels also decreased rapidly as the temperature rose from 20 to 25°C. The expression levels of pelA, algD, and pslA encoding Pel, alginate, and Psl, respectively, were also dramatically affected by temperature, with pelA being regulated in a pattern similar to that of the intracellular c-di-GMP levels, and the pattern seen for algD regulation was the most similar to the actual biofilm formation pattern. Total exopolysaccharide production was thermoregulated and followed the regulation pattern of c-di-GMP. Interestingly, the thermoregulation patterns in biofilm formation were different depending on the strain of P. aeruginosa Unlike PAO1, another strain, PA14, showed a gradual decrease in biofilm formation and c-di-GMP in the range of 20 to 37°C, and P. aeruginosa clinical isolates also showed slightly different patterns in biofilm formation in conjunction with temperature change, suggesting that different strains may sense different temperature ranges for biofilm formation. However, it is obvious that P. aeruginosa forms more biofilms at lower temperatures and that temperature is an important factor in determining the biofilm formation.IMPORTANCE Biofilm formation is an important protection mechanism used by most microorganisms and provides cells with many advantages, like high infectivity, antibiotic resistance, and strong survivability. Since most persistent bacterial infections are believed to be associated with biofilms, biofilm control is an important issue in medicine, environmental engineering, and industry. Biofilm formation is influenced by various environmental factors. Temperature is the most direct environmental cue encountered by microorganisms. Here, we investigated the effect of temperature on the biofilm formation of P. aeruginosa, a notorious pathogen, and found that temperature is an important factor determining the amount and structure of biofilms. Low temperatures greatly increase biofilm formation and give biofilms a highly conspicuous structure. Although thermoregulation of biofilm formation is mainly mediated by c-di-GMP, some c-di-GMP-independent regulations were also observed. This study shows how biofilms are formed at various temperatures and provides new insights to control biofilms using temperature.

Alternative methylation of intron motifs is associated with cancer-related gene expression in both canine mammary tumor and human breast cancer.

BACKGROUND: Canine mammary tumor (CMT) has long been considered as a good animal model for human breast cancer (HBC) due to their pathological and biological similarities. However, only a few aspects of the epigenome have been explored in both HBC and CMT. Moreover, DNA methylation studies have mainly been limited to the promoter regions of genes. RESULTS: Genome-wide methylation analysis was performed in CMT and adjacent normal tissues and focused on the intron regions as potential targets for epigenetic regulation. As expected, many tumor suppressors and oncogenes were identified. Of note, most cancer-associated biological processes were enriched in differentially methylated genes (DMGs) that included intron DMRs (differentially methylated regions). Interestingly, two PAX motifs, PAX5 (tumor suppressive) and PAX6 (oncogenic), were frequently found in hyper- and hypomethylated intron DMRs, respectively. Hypermethylation at the PAX5 motifs in the intron regions of CDH5 and LRIG1 genes were found to be anti-correlated with gene expression, while CDH2 and ADAM19 genes harboring hypomethylated PAX6 motifs in their intron region were upregulated. These results were validated from the specimens originally MBD-sequenced as well as additional clinical samples. We also comparatively investigated the intron methylation and downstream gene expression of these genes using human breast invasive carcinoma (BRCA) datasets in TCGA (The Cancer Genome Atlas) public database. Regional alteration of methylation was conserved in the corresponding intron regions and, consequently, gene expression was also altered in HBC. CONCLUSIONS: This study provides good evidence for the conservation of epigenetic regulation in CMT and HBC, and suggests that intronic methylation can be an important factor in better understanding gene regulation in both CMT and HBC.

Somatic Mutation of PIK3CA (H1047R) Is a Common Driver Mutation Hotspot in Canine Mammary Tumors as Well as Human Breast Cancers.

Breast cancer is one of the most frequently diagnosed cancers in both women and female dogs. Genome-wide association studies in human breast cancer (HBC) have identified hundreds of genetic variations and somatic driver mutations. However, only a handful of variants have been studied for rare HBC and their associations remain inconclusive. Spontaneous canine mammary tumor (CMT) is a great model for HBC, with clinical similarity. We thus performed whole-exome sequencing in 20 pairs of CMT and normal tissues in dogs. We newly found that PIK3CA was the most frequently mutated gene in CMT (45%). Furthermore, canine PIK3CA A3140G (H1047R), at what is known as the mutational hotspot of HBC, is also a hotspot in CMT. Targeted sequencing confirmed that 29% of CMTs had the same PIK3CA A3140G mutation. Integration of the transcriptome suggests that the PIK3CA (H1047R) induced cell metabolism and cell cycle via an increase of PCK2 and a decrease of CDKN1B but had no effect on cell apoptosis. We identified additional significantly mutated genes, including SCRN1 and CLHC1, which have not been reported in HBC. Our study recapitulated some known HBC-associated genes and human cancer signatures in CMT, and identified novel genes that may be relevant to HBC. This study may allow us to better understand both HBC and CMT and lend new insights into the development of biomarkers.

Upregulation of Complement Factor H by SOCS-1/3⁻STAT4 in Lung Cancer.

Complement factor H (CFH) is a fluid phase regulator of complement proteins and functions to prevent complement attack and immune surveillance. CFH is known to inactivate therapeutic antibody-dependent complement-mediated cellular cytotoxicity. We found that CFH was highly expressed in human lung cancer cells and tissues. To investigate mechanisms of CFH upregulation, we searched for a CFH transcription factor and its regulatory factors. First, signal transducer and activator of transcription 4 (STAT4) expression patterns coincided with CFH expression patterns in lung cancer tissues. Knockdown of STAT4 led to decreased CFH secretion from lung cancer cells. STAT4 bound directly to the CFH promoter, as demonstrated by luciferase reporter assay, electrophoretic mobility shift assay (EMSA), and chromatin immunoprecipitation (ChIP) assay, suggesting that STAT4 is a transcription factor for CFH. In addition, a low level of suppressors of cytokine signaling (SOCS)-1/3, a Janus kinase (JAK) inhibitor, was observed in lung cancer cells and its transfection decreased CFH protein levels and promoter activity. Unexpectedly, the low level of SOCS-1/3 was not due to epigenetic silencing. Instead, differential methylation was found on the regulatory region of STAT4 between normal and lung cancer cells. In conclusion, our results demonstrated that CFH is upregulated by constitutive activation of STAT4, which is accounted for by SOCS silencing in lung cancer cells.

Antibiofilm effect of biofilm-dispersing agents on clinical isolates of Pseudomonas aeruginosa with various biofilm structures.

Pseudomonas aeruginosa, an opportunistic human pathogen, causes many biofilm-mediated chronic infections. In this study, biofilm structures of various clinical strains of P. aeruginosa isolated from hospitalized patients were examined and their influence on the biofilm-dispersing effects of chemicals was investigated. The clinical isolates formed structurally distinct biofilms that could be classified into three different groups: 1) mushroom-like, 2) thin flat, and 3) thick flat structures. A dispersion of these differently structured biofilms was induced using two biofilm-dispersing agents, anthranilate and sodium nitroprusside (SNP). Although both SNP and anthranilate could disperse all types of biofilms, the thick flat biofilms were dispersed less efficiently than the biofilms of other structures. This suggests that biofilm-dispersing agents have higher potency on the biofilms of porous structures than on densely packed biofilms.