Mediator complex (MED) 7: a biomarker associated with good prognosis in invasive breast cancer, especially ER+ luminal subtypes

Background: Mediator complex (MED) proteins have a key role in transcriptional regulation, some interacting with the oestrogen receptor (ER). Interrogation of the METABRIC cohort suggested that MED7 may regulate lymphovascular invasion (LVI). Thus MED7 expression was assessed in large breast cancer (BC) cohorts to determine clinicopathological significance. Methods: MED7 gene expression was investigated in the METABRIC cohort (n = 1980) and externally validated using bc-GenExMiner v4.0. Immunohistochemical expression was assessed in the Nottingham primary BC series (n = 1280). Associations with clinicopathological variables and patient outcome were evaluated. Results: High MED7 mRNA and protein expression was associated with good prognostic factors: low grade, smaller tumour size, good NPI, positive hormone receptor status (p < 0.001), and negative LVI (p = 0.04) status. Higher MED7 protein expression was associated with improved BC-specific survival within the whole cohort and ER+/luminal subgroup. Pooled MED7 gene expression data in the external validation cohort confirmed association with better survival, corroborating with the protein expression. On multivariate analysis, MED7 protein was independently predictive of longer BC-specific survival in the whole cohort and Luminal A subtype (p < 0.001). Conclusions: MED7 is an important prognostic marker in BC, particularly in ER+luminal subtypes, associated with improved survival and warrants future functional analysis

Inhibition efficiency of two bipyrazole derivatives on steel corrosion in hydrochloric acid media

244-253The inhibitor effect of two isomers namely 2-(1',5,5'-trimethyl-1H,1'H-3,3'-bipyrazol-1-yl)ethanol (1-TBE) and 2-(1',5,5'-trimethyl-1H,2'H-3,3'-bipyrazol-2-yl)ethanol (2-TBE) on the corrosion of mild steel in 1.0 M hydrochloric acid has been investigated at 308 K using weight loss measurements and electrochemical techniques (impedance spectroscopy and polarisation curves). Inhibition efficiency is dependent upon the pyrazole structure, with 1-TBE serving as a better inhibitor than 2-TBE and its inhibition efficiency increases with the increase of concentration of inhibitor to attain 93% in the presence of 10-3M. Polarisation curves indicate that 1-TBE and 2-TBE act essentially as cathodic inhibitors. Efficiency (E) percent values obtained by various methods are reasonably good in agreement. EIS measurements show an increase of the transfer resistance with the inhibitor concentration. The temperature effect on the corrosion behaviour of steel in 1.0 M HCl without and with the inhibitor at 10-3M is studied in the temperature range 308-333 K, Some thermodynamic parameters such as adsorption heat (H°), adsorption entropy (S°) and adsorption free energy (G°) have been calculated by employing thermodynamic equations. Kinetic parameters such as apparent activation energy and pre-exponential factor have also been calculated. Adsorption of 1-TBE on the mild steel surface in 1.0 M HCl follows the Langmuir isotherm model

Tuning Hsf1 levels drives distinct fungal morphogenetic programs with depletion impairing Hsp90 function and overexpression expanding the target space

<div><p>The capacity to respond to temperature fluctuations is critical for microorganisms to survive within mammalian hosts, and temperature modulates virulence traits of diverse pathogens. One key temperature-dependent virulence trait of the fungal pathogen <i>Candida albicans</i> is its ability to transition from yeast to filamentous growth, which is induced by environmental cues at host physiological temperature. A key regulator of temperature-dependent morphogenesis is the molecular chaperone Hsp90, which has complex functional relationships with the transcription factor Hsf1. Although Hsf1 controls global transcriptional remodeling in response to heat shock, its impact on morphogenesis remains unknown. Here, we establish an intriguing paradigm whereby overexpression or depletion of <i>C</i>. <i>albicans HSF1</i> induces morphogenesis in the absence of external cues. <i>HSF1</i> depletion compromises Hsp90 function, thereby driving filamentation. <i>HSF1</i> overexpression does not impact Hsp90 function, but rather induces a dose-dependent expansion of Hsf1 direct targets that drives overexpression of positive regulators of filamentation, including Brg1 and Ume6, thereby bypassing the requirement for elevated temperature during morphogenesis. This work provides new insight into Hsf1-mediated environmentally contingent transcriptional control, implicates Hsf1 in regulation of a key virulence trait, and highlights fascinating biology whereby either overexpression or depletion of a single cellular regulator induces a profound developmental transition.</p></div

Global gene deletion analysis exploring yeast filamentous growth

The dimorphic switch from a single-cell budding yeast to a filamentous form enables Saccharomyces cerevisiae to forage for nutrients and the opportunistic pathogen Candida albicans to invade human tissues and evade the immune system. We constructed a genome-wide set of targeted deletion alleles and introduced them into a filamentous S. cerevisiae strain, S1278b. We identified genes involved in morphologically distinct forms of filamentation: haploid invasive growth, biofilm formation, and diploid pseudohyphal growth. Unique genes appear to underlie each program, but we also found core genes with general roles in filamentous growth, including MFG1 (YDL233w), whose product binds two morphogenetic transcription factors, Flo8 and Mss11, and functions as a critical transcriptional regulator of filamentous growth in both S. cerevisiae and C. albicans.Peer reviewed: YesNRC publication: Ye

<i>HSF1</i> depletion induces filamentation independently of changes in <i>HSP90</i> expression.

<p>Strains were grown in the absence or presence of 20 μg/mL DOX in rich medium at 30°C. a) A strain was engineered where <i>HSP90</i> is under the control of a constitutive promoter, <i>ACT1p</i>, in the <i>tetO-HSF1/hsf1Δ</i> background. In the <i>ACT1p-HSP90</i> strain, <i>HSF1</i> levels were depleted (left panel) with DOX without altering <i>HSP90</i> expression (right panel). <i>HSF1</i> and <i>HSP90</i> transcript levels were normalized to <i>ACT1</i> and <i>GPD1</i>. Data are means +/- standard error of the means for triplicate samples. ** indicates P value <0.01, ns indicates no significant difference, unpaired t test. b) <i>HSF1</i> depletion induces filamentation independently of changes in <i>HSP90</i> expression.</p

<i>HSF1</i> overexpression induces filamentation independently of changes in <i>HSP90</i> transcript or protein levels, and function.

<p>a) To monitor the effects of <i>HSF1</i> overexpression on filamentation independently of changes in <i>HSP90</i> expression, we engineered strains where <i>HSP90</i> is under the control of a constitutive promoter, <i>ACT1p</i>, in the <i>tetO-HSF1/tetO-HSF1</i> strain. <i>HSF1</i> and <i>HSP90</i> transcript levels were normalized to <i>ACT1</i> and <i>GPD1</i>. Data are means +/- standard error of the means for triplicate samples. In the <i>ACT1p-HSP90</i> strain, <i>HSF1</i> levels are overexpressed (left panel) but <i>HSP90</i> levels do not differ substantially from the wild-type levels (right panel). * indicates P value <0.05, ns indicates no significant difference, unpaired t test. b) Western blot analysis shows that overexpression of <i>HSF1</i> does not impact the levels of Hsp90 protein. Tubulin serves as loading control. c) <i>HSF1</i> overexpression induces filamentation independently of changes in <i>HSP90</i> expression. Strains were grown in the absence of DOX at 30°C. d) Western blot analysis was performed to assay if <i>HSF1</i> overexpression compromises Hsp90 function by monitoring the Hsp90 client protein Hog1. Cells were treated with 5 mM hydrogen peroxide (H₂O₂) for 10 minutes to induce oxidative stress before protein extraction. Overexpression of <i>HSF1</i> does not affect the stability of Hog1, nor does it block its activation. Tubulin serves as a loading control. e) <i>HSF1</i> overexpression does not cause hypersensitivity to the Hsp90 inhibitor geldanamycin (Gda). Growth curves were generated by measuring the optical density of cells grown in the absence and presence of 20 μg/mL DOX in the presence of 3.13 μM geldanamycin. Optical density measurements at 595 nm were taken every 15 minutes with a TECAN plate reader. Experiment was performed in biological quadruplicate, with one representative graph shown.</p

<i>HSF1</i> overexpression and depletion induce filaments that differ in their morphology, nuclear content and dependence on Ras1.

<p>a) Reduced temperature blocks filamentation in response to <i>HSF1</i> overexpression but not in response to <i>HSF1</i> depletion. Strains were grown in the absence or presence of high levels of DOX (20 μg/mL) at the indicated temperature. b) Filaments induced by <i>HSF1</i> overexpression and depletion have distinct features. Cell walls and septa were visualized using calcofluor white and the nuclei were visualized using a strain with the nucleolar protein, Nop1, GFP tagged. c) Filaments induced by <i>HSF1</i> depletion have a significant increase in the percentage of cells that are multinucleate compared to filaments induced by <i>HSF1</i> overexpression. The number of nuclei in at least 300 cells were counted for each condition, for two biological replicates. Means are graphed with the error bars displaying the standard error of means. Unpaired t-test indicates a significant difference in percentage of multinucleate cells, P value is 0.0056. d) Filaments induced by <i>HSF1</i> depletion, but not overexpression, are dependent on the GTPase Ras1. Strains were grown in rich medium with 80 mg/L uridine added, in the presence of no DOX, 0.1 μg/mL DOX (Low DOX), or 20 μg/mL DOX (High DOX) at 30°C.</p

Hsf1 binds to an expanded set of taget genes upon overexpression, inducing the expression of positive regulators of filamentation.

<p>a) ChIP-seq was performed to determine the targets of Hsf1 in strains expressing basal (<i>HSF1-TAP/HSF1</i>) and overexpressed (<i>tetO-HSF1-TAP/tetO-HSF1</i>) levels of Hsf1, which identified an expansion of Hsf1 targets upon overexpression. Genes previously identified as Hsf1 targets and genes involved in <i>C</i>. <i>albicans</i> filamenation are indicated. b) Hsf1 signals at the summit of Hsf1 ChIP-seq peaks in wild-type (basal conditions, top) and <i>HSF1</i> overexpression (bottom) strains. Weak Hsf1 ChIP-seq signal can be seen at the overexpression-specific target sites under basal conditions, suggesting that Hsf1 also binds these sites under basal conditions albeit at a lower level. Legend shows Hsf1 ChIP-seq signal across a 2 kb window spanning Hsf1 binding summits identified by MACS analysis. c) Both basal and <i>HSF1</i> overexpression-specific targets contain the conserved heat shock element (HSE) binding sites. The percentage of ChIP-seq peaks with the HSE motifs TTCnnGAA, TTCn⁷TTC, TTCnnGAAnnTTC, or variations of the motifs termed triple cis motifs (GAA n^0-3 GAA n^0-3 GAA), triple trans motifs (GAA n^0-3 TTC n^0-3 GAA) or triple trans/cis motifs (TTC n^0-3 GAA n^0-3 GAA, GAA n^0-3 GAA n^0-3 TTC, TTC n^0-3 TTC n^0-3 GAA, GAA n^0-3 TTC n^0-3 TTC) are shown for basal and overexpression-specific targets. d) MA plot showing difference in gene expression between wild-type and <i>HSF1</i> overexpression strains. Genes more than 1.5-fold up-regulated in the <i>HSF1</i> overexpression strain as compared to the wild-type strain are shown in red, whereas genes more than 1.5-fold down-regulated are shown in blue. Genes of interest based on their roles in filamentation are indicated. <i>HSF1</i> is highlighted in purple. e) A Euler diagram depicting the overlap between differentially regulated genes upon <i>HSF1</i> overexpression determined by RNA-seq and Hsf1 bound targets determined by ChIP-seq. Hsf1 targets includes those promoters bound in either the wild-type or overexpression strain. Selected genes associated with filamentation are indicated in the diagram. f) A heat map showing gene expression changes upon <i>HSF1</i> overexpression for filamentation regulators. Colored dots are included to indicate whether the respective gene is bound by Hsf1 in the wild-type strain (in red) or only when Hsf1 is overexpressed (in orange). The colour bar depicts the change in expression as determined by RNA-seq.</p

<i>HSF1</i> depletion induces filamentation by compromising Hsp90 function, independently of changes in <i>HSP90</i> expression.

<p>a) Western blot analysis was performed to assay if <i>HSF1</i> depletion compromises Hsp90 function by monitoring the Hsp90 client protein Hog1. Strains were grown in the absence or presence of 20 μg/mL DOX. Cells were treated with 5 mM hydrogen peroxide (H₂O₂) for 10 minutes to induce oxidative stress before protein extraction. Depletion of Hsf1 reduces the levels of phosphorylated Hog1 (pHog1) but not total Hog1 levels, even in the isogenic strain with constitutive <i>HSP90</i> expression. Tubulin levels serve as loading control. WT indicates the wild type control. Experiment was performed in biological quadruplicate and a representative image is shown, with the western blots for the additional replicates shown in <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007270#pgen.1007270.s015" target="_blank">S8 Fig</a>.</b> b) <i>HSF1</i> depletion causes a hypersensitivity to the Hsp90 inhibitor geldanamycin, even when <i>HSP90</i> expression is constitutive and independent of Hsf1. Growth curves were generated by measuring the optical density of cells grown in the absence or presence of 20 μg/mL DOX in the presence of a concentration of geldanamycin that does not inhibit the growth of the wild-type strain (3.13 μM). Optical density at 595 nm was measured every 15 minutes with a TECAN plate reader grown with high orbital shaking at 30°C. Experiment was performed in biological quadruplicate, with one representative graph shown. c) <i>HSF1</i> depletion causes filamentation at lower concentrations of geldanamycin (Gda) than is necessary to induce filamentation of the wild-type strain. Strains were grown in static conditions in the presence of no DOX or 20 μg/mL DOX, and in the presence of no geldanamycin (Gda) or 3.13 μM geldanamycin (Gda) at 30°C. In static growth conditions, 3.13 μM geldanamycin does not inhibit growth of the strains.</p