FOXO1 promotes the expression of canonical WNT target genes in examined basal-like breast and glioblastoma multiforme cancer cells.

Basal-like breast cancer (BBC) and glioblastoma multiforme (GBM) are aggressive cancers associated with poor prognosis. BBC and GBM have stem cell-like gene expression signatures, which are in part driven by forkhead box O (FOXO) transcription factors. To gain further insight into the impact of FOXO1 in BBC, we treated BT549 cells with AS1842856 and performed RNA sequencing. AS1842856 binds to unphosphorylated FOXO1 and inhibits its ability to directly bind to DNA. Gene Set Enrichment Analysis indicated that a set of WNT pathway target genes, including lymphoid enhancer-binding factor 1 (LEF1) and transcription factor 7 (TCF7), were robustly induced after AS1842856 treatment. These same genes were also induced in GBM cell lines U87MG, LN18, LN229, A172, and DBTRG upon AS1842856 treatment. By contrast, follow-up RNA interference (RNAi) targeting of FOXO1 led to reduced LEF1 and TCF7 gene expression in BT549 and U87MG cells. In agreement with RNAi experiments, CRISPR Cas9-mediated FOXO1 disruption reduced the expression of canonical WNT genes LEF1 and TCF7 in U87MG cells. The loss of TCF7 gene expression in FOXO1 disruption mutants was restored by exogenous expression of the DNA-binding-deficient FOXO1-H215R. Therefore, FOXO1 induces TCF7 in a DNA-binding-independent manner, similar to other published FOXO1-activated genes such as TCF4 and hes family bHLH transcription factor 1. Our work demonstrates that FOXO1 promotes canonical WNT gene expression in examined BBC and GBM cells, similar to results found in Drosophila melanogaster, T-cell development, and murine acute myeloid leukemia models.

NVP-BEZ235 or JAKi Treatment leads to decreased survival of examined GBM and BBC cells.

Cancer cells almost universally harbor constitutively active Phosphatidylinositol-3 Kinase (PI3K) Pathway activity via mutation of key signaling components and/or epigenetic mechanisms. Scores of PI3K Pathway inhibitors are currently under investigation as putative chemotherapeutics. However, feedback and stem cell mechanisms induced by PI3K Pathway inhibition can lead to reduced treatment efficacy. To address therapeutic barriers, we examined whether JAKi would reduce stem gene expression in a setting of PI3K Pathway inhibition in order to improve treatment efficacy. We targeted the PI3K Pathway with NVP-BEZ235 (dual PI3K and mTOR inhibitor) in combination with the Janus Kinase inhibitor JAKi in glioblastoma (GBM) and basal-like breast cancer (BBC) cell lines. We examined growth, gene expression, and apoptosis in cells treated with NVP-BEZ235 and/or JAKi. Growth and recovery assays showed no significant impact of dual treatment with NVP-BEZ235/JAKi compared to NVP-BEZ235 treatment alone. Gene expression and flow cytometry revealed that single and dual treatments induced apoptosis. Stem gene expression was retained in dual NVP-BEZ235/JAKi treatment samples. Future in vivo studies may give further insight into the impact of combined NVP-BEZ235/JAKi treatment in GBM and BBC.

The PI3K pathway impacts stem gene expression in a set of glioblastoma cell lines.

BACKGROUND: The PI3K pathway controls diverse cellular processes including growth, survival, metabolism, and apoptosis. Nuclear FOXO factors were observed in cancers that harbor constitutively active PI3K pathway output and stem signatures. FOXO1 and FOXO3 were previously published to induce stem genes such as OCT4 in embryonic stem cells. Here, we investigated FOXO-driven stem gene expression in U87MG glioblastoma cells. METHODS: PI3K-activated cancer cell lines were investigated for changes in gene expression, signal transduction, and clonogenicity under conditions with FOXO3 disruption or exogenous expression. The impact of PI3K pathway inhibition on stem gene expression was examined in a set of glioblastoma cell lines. RESULTS: We found that CRISPR-Cas9-mediated FOXO3 disruption in U87MG cells caused decreased OCT4 and SOX2 gene expression, STAT3 phosphorylation on tyrosine 705 and clonogenicity. FOXO3 over expression led to increased OCT4 in numerous glioblastoma cancer cell lines. Strikingly, treatment of glioblastoma cells with NVP-BEZ235 (a dual inhibitor of PI3K and mTOR), which activates FOXO factors, led to robust increases OCT4 gene expression. Direct FOXO factor recruitment to the OCT4 promoter was detected by chromatin immunoprecipitation analyses using U87MG extracts. DISCUSSION: We show for the first time that FOXO transcription factors promote stem gene expression glioblastoma cells. Treatment with PI3K inhibitor NVP-BEZ235 led to dramatic increases in stem genes in a set of glioblastoma cell lines. CONCLUSION: Given that, PI3K inhibitors are actively investigated as targeted cancer therapies, the FOXO-mediated induction of stem genes observed in this study highlights a potential hazard to PI3K inhibition. Understanding the molecular underpinnings of stem signatures in cancer will allow refinements to therapeutic strategies. Targeting FOXO factors to reduce stem cell characteristics in concert with PI3K inhibition may prove therapeutically efficacious.

Seasonal detection of atrazine and atzA in man-made waterways receiving agricultural runoff in a subtropical, semi-arid environment (Hidalgo County, Texas, USA).

Atrazine is a widely-used herbicide that can impact non-target organisms in the environment but can be biologically degraded by several types of microorganisms. In this study, the gene atzA, which encodes for the initial step in bacterially-mediated atrazine degradation, was used as an indicator of atrazine pollution in agricultural canals located in Hidalgo County, Texas, USA. The concentration of atrazine and atzA were monitored once per month for 12 months during 2010-2011. Atrazine was measured using an enzyme-linked immunosorbent assay; atzA abundance was monitored using Quantitative Polymerase Chain Reaction (Q-PCR) analyses. Abundance of atrazine and atzA were compared with rainy versus dry months and during planting versus non-planting months. Results showed that atrazine levels varied from below detection to 0.43 ppb and were not influenced by precipitation or planting season. Concentrations of the gene atzA were significantly different in rainy versus dry months; during planting versus non-planting times of the year; and in the interaction of precipitation and planting season. The highest concentration of atzA, approx. 4.57 × 108 gene copies ml-1, was detected in July 2010-a rainy, planting month in Hidalgo County, South Texas. However, atrazine was below detection during that month. We conclude that Q-PCR using atzA as an indicator gene is a potential method for monitoring low levels of atrazine pollution in environmental samples.

A CRISPR View of Biological Mechanisms.

A decade ago, only six manuscripts would be found on a PubMed search for "CRISPR," compared to 2,011 manuscripts in 2016. The purpose of this review is to discuss this emergent technology that has revolutionized molecular biological research in just a few years. Endogenous CRISPR mechanisms are harbored by bacteria and archaea as an adaptive defense system that targets foreign DNA from viruses and plasmids. CRISPR has been adapted as a genome editing tool in a plethora of organisms ranging from yeast to humans. This tool has been employed to create loss of function mutations, gain of function mutations, and tagged alleles in a wide range of settings. CRISPR is now extensively employed for genetic screens. CRISPR has also been adapted to study transcriptional regulation. This versatile and relatively facile technique has, and will be, tremendously impactful in research areas such as biomedical sciences, agriculture, and the basic sciences.

New Frontiers for the NFIL3 bZIP Transcription Factor in Cancer, Metabolism and Beyond.

The bZIP transcription factor NFIL3 (Nuclear factor Interleukin 3 regulated, also known as E4 binding protein 4, E4BP4) regulates diverse biological processes from circadian rhythm to cellular viability. Recently, a host of novel roles have been identified for NFIL3 in immunological signal transduction, cancer, aging and metabolism. Elucidating the signaling pathways that are impacted by NFIL3 and the regulatory mechanisms that it targets, inhibits or activates will be critical for developing a clearer picture of its physiological roles in disease and normal processes. This review will discuss the recent advances and emerging issues regarding NFIL3-mediated transcriptional regulation of CEBPß and FOXO1 activated genes and signal transduction.

Nickel and cobalt resistance engineered in Escherichia coli by overexpression of serine acetyltransferase from the nickel hyperaccumulator plant Thlaspi goesingense.

The overexpression of serine acetyltransferase from the Ni-hyperaccumulating plant Thlaspi goesingense causes enhanced nickel and cobalt resistance in Escherichia coli. Furthermore, overexpression of T. goesingense serine acetyltransferase results in enhanced sensitivity to cadmium and has no significant effect on resistance to zinc. Enhanced nickel resistance is directly related to the constitutive overactivation of sulfur assimilation and glutathione biosynthesis, driven by the overproduction of O-acetyl-L-serine, the product of serine acetyltransferase and a positive regulator of the cysteine regulon. Nickel in the serine acetyltransferase-overexpressing strains is not detoxified by coordination or precipitation with sulfur, suggesting that glutathione is involved in reducing the oxidative damage imposed by nickel.

The plant CDF family member TgMTP1 from the Ni/Zn hyperaccumulator Thlaspi goesingense acts to enhance efflux of Zn at the plasma membrane when expressed in Saccharomyces cerevisiae.

To avoid metal toxicity, organisms have evolved mechanisms including efflux of metal ions from cells and sequestration into internal cellular compartments. Members of the ubiquitous cation diffusion facilitator (CDF) family are known to play an important role in these processes. Overexpression of the plant CDF family member metal tolerance protein 1 (MTP1) from the Ni/Zn hyperaccumulator Thlaspi goesingense (TgMTP1), in the Saccharomyces cerevisiaeDelta zinc resistance conferring (zrc)1Delta cobalt transporter (cot)1 double mutant, suppressed the Zn sensitivity of this strain. T. goesingense was found to contain several allelic variants of TgMTP1, all of which confer similar resistance to Zn in Deltazrc1Deltacot1. Similarly, MTP1 from various hyperaccumulator and non-accumulator species also confer similar resistance to Zn. Deltazrc1Deltacot1 lacks the ability to accumulate Zn in the vacuole and has lower accumulation of Zn after either long- or short-term Zn exposure. Expression of TgMTP1 in Deltazrc1Deltacot1 leads to further lowering of Zn accumulation and an increase in Zn efflux from the cells. Expression of TgMTP1 in a V-type ATPase-deficient S. cerevisiae strain also confers increased Zn resistance. In vivo and in vitro immunological staining of hemagglutinin (HA)-tagged TgMTP1::HA reveals the protein to be localized in both the S. cerevisiae vacuolar and plasma membranes. Taken together, these data are consistent with MTP1 functioning to enhance plasma membrane Zn efflux, acting to confer Zn resistance independent of the vacuole in S. cerevisiae. Transient expression in Arabidopsis thaliana protoplasts also reveals that TgMTP1::green fluorescent protein (GFP) is localized at the plasma membrane, suggesting that TgMTP1 may also enhance Zn efflux in plants.

Increased glutathione biosynthesis plays a role in nickel tolerance in thlaspi nickel hyperaccumulators.

Worldwide more than 400 plant species are now known that hyperaccumulate various trace metals (Cd, Co, Cu, Mn, Ni, and Zn), metalloids (As) and nonmetals (Se) in their shoots. Of these, almost one-quarter are Brassicaceae family members, including numerous Thlaspi species that hyperaccumulate Ni up to 3% of there shoot dry weight. We observed that concentrations of glutathione, Cys, and O-acetyl-l-serine (OAS), in shoot tissue, are strongly correlated with the ability to hyperaccumulate Ni in various Thlaspi hyperaccumulators collected from serpentine soils, including Thlaspi goesingense, T. oxyceras, and T. rosulare, and nonaccumulator relatives, including T. perfoliatum, T. arvense, and Arabidopsis thaliana. Further analysis of the Austrian Ni hyperaccumulator T. goesingense revealed that the high concentrations of OAS, Cys, and GSH observed in this hyperaccumulator coincide with constitutively high activity of both serine acetyltransferase (SAT) and glutathione reductase. SAT catalyzes the acetylation of l-Ser to produce OAS, which acts as both a key positive regulator of sulfur assimilation and forms the carbon skeleton for Cys biosynthesis. These changes in Cys and GSH metabolism also coincide with the ability of T. goesingense to both hyperaccumulate Ni and resist its damaging oxidative effects. Overproduction of T. goesingense SAT in the nonaccumulator Brassicaceae family member Arabidopsis was found to cause accumulation of OAS, Cys, and glutathione, mimicking the biochemical changes observed in the Ni hyperaccumulators. In these transgenic Arabidopsis, glutathione concentrations strongly correlate with increased resistance to both the growth inhibitory and oxidative stress induced effects of Ni. Taken together, such evidence supports our conclusion that elevated GSH concentrations, driven by constitutively elevated SAT activity, are involved in conferring tolerance to Ni-induced oxidative stress in Thlaspi Ni hyperaccumulators.

Chemical form and distribution of selenium and sulfur in the selenium hyperaccumulator Astragalus bisulcatus.

In its natural habitat, Astragalus bisulcatus can accumulate up to 0.65% (w/w) selenium (Se) in its shoot dry weight. X-ray absorption spectroscopy has been used to examine the selenium biochemistry of A. bisulcatus. High concentrations of the nonprotein amino acid Se-methylseleno-cysteine (Cys) are present in young leaves of A. bisulcatus, but in more mature leaves, the Se-methylseleno-Cys concentration is lower, and selenate predominates. Seleno-Cys methyltransferase is the enzyme responsible for the biosynthesis of Se-methylseleno-Cys from seleno-Cys and S-methyl-methionine. Seleno-Cys methyltransferase is found to be expressed in A. bisulcatus leaves of all ages, and thus the biosynthesis of Se-methylseleno-Cys in older leaves is limited earlier in the metabolic pathway, probably by an inability to chemically reduce selenate. A comparative study of sulfur (S) and Se in A. bisulcatus using x-ray absorption spectroscopy indicates similar trends for oxidized and reduced Se and S species, but also indicates that the proportions of these differ significantly. These results also indicate that sulfate and selenate reduction are developmentally correlated, and they suggest important differences between S and Se biochemistries.