The Transcription Factors Snail and Slug Activate the Transforming Growth Factor-Beta Signaling Pathway in Breast Cancer

The transcriptional repressors Snail and Slug are situated at the core of several signaling pathways proposed to mediate epithelial to mesenchymal transition or EMT, which has been implicated in tumor metastasis. EMT involves an alteration from an organized, epithelial cell structure to a mesenchymal, invasive and migratory phenotype. In order to obtain a global view of the impact of Snail and Slug expression, we performed a microarray experiment using the MCF-7 breast cancer cell line, which does not express detectable levels of Snail or Slug. MCF-7 cells were infected with Snail, Slug or control adenovirus, and RNA samples isolated at various time points were analyzed across all transcripts. Our analyses indicated that Snail and Slug regulate many genes in common, but also have distinct sets of gene targets. Gene set enrichment analyses indicated that Snail and Slug directed the transcriptome of MCF-7 cells from a luminal towards a more complex pattern that includes many features of the claudin-low breast cancer signature. Of particular interest, genes involved in the TGF-beta signaling pathway are upregulated, while genes responsible for a differentiated morphology are downregulated following Snail or Slug expression. Further we noticed increased histone acetylation at the promoter region of the transforming growth factor beta-receptor II (TGFBR2) gene following Snail or Slug expression. Inhibition of the TGF-beta signaling pathway using selective small-molecule inhibitors following Snail or Slug addition resulted in decreased cell migration with no impact on the repression of cell junction molecules by Snail and Slug. We propose that there are two regulatory modules embedded within EMT: one that involves repression of cell junction molecules, and the other involving cell migration via TGF-beta and/or other pathways

Preparation and characterization of protein-nanotube conjugates

This chapter describes methods of immobilizing proteins on carbon nanotubes, using two different routes—physical adsorption and covalent attachment. We also provide an overview on how such conjugates can be characterized with the help of various techniques, such as Raman, Fourier transform infrared (FT-IR), circular dichroism (CD), and fluorescence spectroscopies, in addition to the standard enzyme kinetic analyses of activity and stability. Both the attachment routes—covalent and noncovalent—could be used to prepare protein conjugates that retained a significant fraction of their native structure and function; furthermore, the protein conjugates were operationally stable, reusable, and functional even under harsh denaturing conditions. These studies therefore corroborate the use of these immobilization methods to engineer functional carbon nanotube-protein hybrids that are highly active and stable

Structure, Function, and Stability of Enzymes Covalently Attached to Single-Walled Carbon Nanotubes

We describe the structure, activity, and stability of enzymes covalently attached to single-walled carbon nanotubes (SWNTs). Conjugates of SWNTs with three functionally unrelated enzymeshorseradish peroxidase, subtilisin Carlsberg, and chicken egg white lysozymewere found to be soluble in aqueous solutions. Furthermore, characterization of the secondary and tertiary structure of the immobilized proteins by circular dichroism and fluorescence spectroscopies, respectively, and determination of enzyme kinetics revealed that the enzymes retained a high fraction of their native structure and activity upon attachment to SWNTs. The SWNT−enzyme conjugates were also more stable in guanidine hydrochloride (GdnHCl) and at elevated temperatures relative to their solution counterparts. Thus, these protein conjugates represent novel preparations that possess the attributes of both soluble enzymeshigh activity and low diffusional resistanceand immobilized enzymeshigh stabilitymaking them attractive choices for applications ranging from diagnostics and sensing to drug delivery

HAfTs are novel lncRNA transcripts from aflatoxin exposure

<div><p>The transcriptome can reveal insights into precancer biology. We recently conducted RNA-Seq analysis on liver RNA from male rats exposed to the carcinogen, aflatoxin B1 (AFB1), for 90 days prior to liver tumor onset. Among >1,000 differentially expressed transcripts, several novel, unannotated Cufflinks-assembled transcripts, or HAfTs (<u><b>H</b></u>epatic <u><b>Af</b></u>latoxin <u><b>T</b></u>ranscripts) were found. We hypothesized PCR-cloning and RACE (rapid amplification of cDNA ends) could further HAfT identification. Sanger data was obtained for 6 transcripts by PCR and 16 transcripts by 5’- and 3’-RACE. BLAST alignments showed, with two exceptions, HAfT transcripts were lncRNAs, >200nt without apparent long open reading frames. Six rat HAfT transcripts were classified as ‘novel’ without RefSeq annotation. Sequence alignment and genomic synteny showed each rat lncRNA had a homologous locus in the mouse genome and over half had homologous loci in the human genome, including at least two loci (and possibly three others) that were previously unannotated. While HAfT functions are not yet clear, coregulatory roles may be possible from their adjacent orientation to known coding genes with altered expression that include 8 HAfT-gene pairs. For example, a unique rat HAfT, homologous to Pvt1, was adjacent to known genes controlling cell proliferation. Additionally, PCR and RACE Sanger sequencing showed many alternative splice variants and refinements of exon sequences compared to Cufflinks assembled transcripts and gene prediction algorithms. Presence of multiple splice variants and short tandem repeats found in some HAfTs may be consequential for secondary structure, transcriptional regulation, and function. In summary, we report novel, differentially expressed lncRNAs after exposure to the genotoxicant, AFB1, prior to neoplastic lesions. Complete cloning and sequencing of such transcripts could pave the way for a new set of sensitive and early prediction markers for chemical hepatocarcinogens.</p></div

Transcriptional pathways linked to fetal and maternal hepatic dysfunction caused by gestational exposure to perfluorooctanoic acid (PFOA) or hexafluoropropylene oxide-dimer acid (HFPO-DA or GenX) in CD-1 mice

Per- and polyfluoroalkyl substances (PFAS) comprise a diverse class of chemicals used in industrial processes, consumer products, and fire-fighting foams which have become environmental pollutants of concern due to their persistence, ubiquity, and associations with adverse human health outcomes, including in pregnant persons and their offspring. Multiple PFAS are associated with adverse liver outcomes in adult humans and toxicological models, but effects on the developing liver are not fully described. Here we performed transcriptomic analyses in the mouse to investigate the molecular mechanisms of hepatic toxicity in the dam and its fetus after exposure to two different PFAS, perfluorooctanoic acid (PFOA) and its replacement, hexafluoropropylene oxide-dimer acid (HFPO-DA, known as GenX). Pregnant CD-1 mice were exposed via oral gavage from embryonic day (E) 1.5–17.5 to PFOA (0, 1, or 5 mg/kg-d) or GenX (0, 2, or 10 mg/kg-d). Maternal and fetal liver RNA was isolated (N = 5 per dose/group) and the transcriptome analyzed by Affymetrix Array. Differentially expressed genes (DEG) and differentially enriched pathways (DEP) were obtained. DEG patterns were similar in maternal liver for 5 mg/kg PFOA, 2 mg/kg GenX, and 10 mg/kg GenX (R2: 0.46–0.66). DEG patterns were similar across all 4 dose groups in fetal liver (R2: 0.59–0.81). There were more DEGs in fetal liver compared to maternal liver at the low doses for both PFOA (fetal = 69, maternal = 8) and GenX (fetal = 154, maternal = 93). Upregulated DEPs identified across all groups included Fatty Acid Metabolism, Peroxisome, Oxidative Phosphorylation, Adipogenesis, and Bile Acid Metabolism. Transcriptome-phenotype correlation analyses demonstrated > 1000 maternal liver DEGs were significantly correlated with maternal relative liver weight (R2 >0.92). These findings show shared biological pathways of liver toxicity for PFOA and GenX in maternal and fetal livers in CD-1 mice. The limited overlap in specific DEGs between the dam and fetus suggests the developing liver responds differently than the adult liver to these chemical stressors. This work helps define mechanisms of hepatic toxicity of two structurally unique PFAS and may help predict latent consequences of developmental exposure

PCR cloning clarifies HAfT6 transcript sequence and structure.

<p>The structure of HAfT 6 was studied by PCR cloning and Sanger sequencing. The overlap of Cufflinks transcripts (Cufflinks_00024116 and 00024274) suggested a longer, more complex transcript. Primer sets were designed using sequences from both Cufflinks transcripts to test if they comprised a longer single transcript. In Panel A, several primer sets spanned different portions of the two Cufflinks transcripts at this locus. Individual PCR products shown in the agarose gel were excised separately, cloned and Sanger sequenced. Primer Set#7 was amplified twice to clearly show a PCR product (far right lane). In Panel B, the combined consensus Sanger sequences from all primer sets showed two variants, X1 and X2, containing either four or five exons, respectively. Note that red bands in black exons indicate Sanger sequence base variants that differ from alignment with Rn6. See text for further details.</p

Summary of NCBI search alignments of HAfT consensus sequences.

<p>Summary of NCBI search alignments of HAfT consensus sequences.</p

Novel HAfTs.

<p>Four novel, unannotated HAfTs and variants are shown in the UCSC genome browser. AFB1 treatment resulted in an increased number of RNA-Seq reads at specific genomic loci that enabled assembly of Cufflinks transcripts. Primers were designed from Cufflinks transcripts and PCR produced amplicons for portions of HAfT19 and 20 in Panel A, and HAfT22 and 24 in Panel B. Additional variants may exist for these novel HAfT transcripts.</p

Workflow for PCR and RACE, cloning and sequencing.

<p>See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190992#sec002" target="_blank">Methods</a> for further details.</p

Synteny of HAfTs in mouse and human chromosomes^a.

<p>Synteny of HAfTs in mouse and human chromosomes<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190992#t002fn001" target="_blank">^a</a>.</p