Heat Shock Factor Protein Research Articles

PSIII-3 Association of heat shock factor protein 1 and prolactin receptor variants with molecular breeding values in Senepol cattle

Abstract Thermal stress has been associated with lower food intake and growth rate in beef cattle. The purpose of this study was to assess the segregation of the p.Leu462* nonsense mutation in the prolactin receptor gene (PRLR) responsible for slick hair (SH) phenotype in Senepol and a T >C missense variant (V344A) located in the heat shock transcription factor protein 1 gene (HSF1). We compared the potential association of these polymorphisms with molecular breeding values (MBV) that were estimated using a panel of commercial genetic markers (Igenity, Neogen Corp). For this, 29 females and 21 males were genotyped by PCR and sequencing. Three genotypes were observed for the p.Leu462* with frequencies of 0.02 C/C, 0.16 C/- and 0.82 -/-. For V344A only two genotypes were observed with frequencies of 0.86/TT and 0.14/TC. This polymorphism was in linkage disequilibrium with a C >T synonymous variant; therefore, for the statistical analyses, the HSF1 genotypes were combined as TT/CC and TC/CT. The GLIMMIX procedure and Tukey test of SAS were used (effect of sex, HSF1 and SH genotype and interactions). MBV for production traits such as average daily gain, tenderness, yearling weight (YW), marbling, fat thickness (FT), percent choice (PC), ribeye area (RA) and carcass weight. Effect of sex (P < .05) were found for YW (males: 6.93lbs ± 0.53, female: 5.74lbs ± 0.57) and for PC (female: 16.17±0.75, male: 15.02±0.55). Differences between HSF1 genotype combinations were significant for the MBV of RA (TC/CT: 0.96in2±0.10; TT/CC: 0.781in2±0.07 and for FT (TC/CT: 0.16in±0.02; TTCC: 0.12in±0.01). The similarity in segregation of SH responsible genotypes (C/- and -/-) and HSF1 (TC/CT and TT/CC) in Senepol could indicate a potential role of HSF1 in adaptation to heat stress in cattle with an additional effect in muscling and fat deposition.

Heat Shock Factor 1 in Relation to Tumor Angiogenesis and Disease Progression in Patients With Pancreatic Cancer.

In this article, the aims were to study the expression of heat shock factor 1 (HSF1) in patients with pancreatic cancer and to elucidate the relevance between HSF1, angiogenesis, clinicopathological factors, and prognosis. Pancreatic cancer, paracancerous, and normal pancreatic tissues were collected. The HSF1 RNA and protein expressions were identified using quantitative real-time reverse transcription polymerase chain reaction and immunohistochemical staining. Associations of HSF1 and cluster of differentiation 34 with clinical variables and disease outcomes were investigated. Compared with the normal pancreatic and paracancerous tissue, HSF1 RNA and protein significantly showed higher expression in the pancreatic cancer tissue and was significantly associated with microvessel density. The high expression of HSF1 did not correspond to the patients' sex, age, carcinoembryonic antigen level, diameter of tumors, and locations; however, it corresponded significantly with carbohydrate antigen 19-9 level, lymph node metastasis, tumor node metastasis stage, differentiation degree, vascular invasion, and distant metastasis. The expression levels of HSF1 and cluster of differentiation 34 were significantly correlated with prognosis, disease specificity, and survival. The high expression of HSF1 would lead to worse prognosis and decrease in survival time and disease-free survival time. HSF1 expression level in pancreatic cancer tissue could be an ideal prognostic biomarker for risk stratification and a potential therapeutic target for patients with pancreatic cancer.

CYCLIC NUCLEOTIDE-GATED ION CHANNELs 14 and 16 Promote Tolerance to Heat and Chilling in Rice.

Calcium signaling has been postulated to be critical for both heat and chilling tolerance in plants, but its molecular mechanisms are not fully understood. Here, we investigated the function of two closely related cyclic nucleotide-gated ion channel (CNGC) proteins, OsCNGC14 and OsCNGC16, in temperature-stress tolerance in rice (Oryza sativa) by examining their loss-of-function mutants generated by genome editing. Under both heat and chilling stress, both the cngc14 and cngc16 mutants displayed reduced survival rates, higher accumulation levels of hydrogen peroxide, and increased cell death. In the cngc16 mutant, the extent to which some genes were induced and repressed in response to heat stress was altered and some Heat Shock factor (HSF) and Heat Shock Protein (HSP) genes were slightly more induced compared to the wild type. Furthermore, the loss of either OsCNGC14 or OsCNGC16 reduced or abolished cytosolic calcium signals induced by either heat or chilling stress. Therefore, OsCNGC14 and OsCNGC16 are required for heat and chilling tolerance and are modulators of calcium signals in response to temperature stress. In addition, loss of their homologs AtCNGC2 and AtCNGC4 in Arabidopsis (Arabidopsis thaliana) also led to compromised tolerance of low temperature. Thus, this study indicates a critical role of CNGC genes in both chilling and heat tolerance in plants, suggesting a potential overlap in calcium signaling in response to high- and low-temperature stress.

Evaluation of activation of HSF and UPR protein folding pathways by chemical stressors using a yeast reporter system for a hypothesis driven laboratory exercise

Heat shock factor (HSF) is a transcription factor that is activated in response to an accumulation of misfolded proteins in the cytoplasm. In contrast, the unfolded protein response (UPR) is a transcriptional response pathway that responds to the accumulation of misfolded proteins in the endoplasmic reticulum. Both pathways, when activated by misfolded proteins, result in a transcriptional response that helps the cell to survive by making factors to help the cell sequester and destroy the misfolded proteins. Using aseptic techniques, Saccharomyces cerevisiae with UPR or HSF Beta‐galactosidase reporter plasmids were grown and tested with various doses of 4 different chemical stressors (Dithiothreitol, Diamide, Hydrogen Peroxide, Beta‐Mercaptoethanol) that may induce protein misfolding. Beta‐galactosidase assays were completed to determine the transcriptional response of the HSF and UPR pathways to the chemical stressors. Importantly, this activity emphasizes the mechanism of action of two different cell signaling pathway and demonstrates the use of a marker gene to determine transcriptional activity of a pathway. This laboratory exercises engages the student in critical thinking and data analysis skills as they evaluate data to determine if their chemical stressor activated both pathways or only one pathway.

Comparative modeling and mutual docking of structurally uncharacterized heat shock protein 70 and heat shock factor-1 proteins in water buffalo

Aim:In this study, a wide range of in silico investigation of Bubalus bubalis (BB) heat shock protein 70 (HSP70) and heat shock factor-1 (HSF1) has been performed, ranging from sequence evaluation among species to homology modeling along with their docking studies to decipher the interacting residues of both molecules.Materials and Methods:Protein sequences of BB HSP70 and HSF1 were retrieved from NCBI database in FASTA format. Primary and secondary structure prediction were computed using Expasy ProtParam server and Phyre2 server, respectively. TMHMM server was used to identify the transmembrane regions in HSP70. Multiple sequence alignment and comparative analysis of the protein was carried out using MAFFT and visualization was created using ESPript 3.0. Phylogenetic analysis was accomplished by COBALT. Interactions of HSP70 with other proteins were studied using STRING database. Modeller 9.18, RaptorX, Swiss-Modeller, Phyre2, and I-TASSER were utilized to design the three-dimensional structure of these proteins followed by refinement; energy minimization was accomplished using ModRefiner and SPDBV program. Stereochemical quality along with the accuracy of the predicted models and their visualization was observed by PROCHECK program of PDBsum and UCSF Chimera, respectively. ClusPro 2.0 server was accessed for the docking of the receptor protein with the ligand.Results:The lower value of Grand Average of Hydropathy indicates the more hydrophilic nature of HSP70 protein. Value of the instability index (II) classified the protein as stable. No transmembrane region was reported for HSP70 by TMHMM server. Phylogenetic analysis based on multiple sequence alignments (MSAs) by COBALT indicated more evolutionarily closeness of Bos indicus (BI) with Bos taurus as compared to BI and BB. STRING database clearly indicates the HSF1 as one of the interacting molecules among 10 interacting partners with HSP 70. The best hit of 3D model of HSP70 protein and HSF1 was retrieved from I-TASSER and Phyre2, respectively. Interacting residues and type of bonding between both the molecules which were docked by ClusPro 2.0 were decoded by PIC server. Hydrophobic interactions, protein-protein main-chain-side-chain hydrogen bonds, and protein-protein side-chain-side-chain hydrogen bonds were delineated in this study.Conclusion:This is the first-ever study on in silico interaction of HSP70 and HSF1 proteins in BB. Several bioinformatics web tools were utilized to study secondary structure along with comparative modeling, physicochemical properties, and protein-protein interaction. The various interacting amino acid residues of both proteins have been indicated in this study.

Multifactorial Attenuation of the Murine Heat Shock Response With Age.

Age-dependent perturbation of the cellular stress response affects proteostasis and other key functions relevant to cellular action and survival. Central to age-related changes in the stress response is loss of heat shock factor 1 (HSF1)-DNA binding and transactivation properties. This report elucidates how age alters different checkpoints of HSF1 activation related to posttranslational modification and protein interactions. When comparing liver extracts from middle aged (12 M) and old (24 M) mice, significant differences are found in HSF1 phosphorylation and acetylation. HSF1 protein levels and messenger RNA decline with age, but its protein levels are stress-inducible and exempt from age-dependent changes. This surprising adaptive change in the stress response has additional implications for aging and chronic physiological stress that might explain an age-dependent dichotomy of HSF1 protein levels that are low in neurodegeneration and elevated in cancer.

Inhibition of Heat Shock Factor 1 Enhances Repressive Molecular Mechanisms on the POMC Promoter

Background: Cushing’s disease (CD) is caused by adrenocorticotropic hormone (ACTH)-secreting pituitary tumours. They express high levels of heat shock protein 90 and heat shock factor 1 (HSF1) in comparison to the normal tissue counterpart, indicating activated cellular stress. Aims: Our objectives were: (1) to correlate HSF1 expression with clinical features and hormonal/radiological findings of CD, and (2) to investigate the effects of HSF1 inhibition as a target for CD treatment. Patients/Methods: We examined the expression of total and pSer³²⁶HSF1 (marker for its transcriptional activation) by Western blot on eight human CD tumours and compared to the HSF1 status of normal pituitary. We screened a cohort of 45 patients with CD for HSF1 by immunohistochemistry and correlated the HSF1 immunoreactivity score with the available clinical data. We evaluated the effects of HSF1 silencing with RNA interference and the HSF1 inhibitor KRIBB11 in AtT-20 cells and four primary cultures of human corticotroph tumours. Results: We show that HSF1 protein is highly expressed and transcriptionally active in CD tumours in comparison to normal pituitary. The immunoreactivity score for HSF1 did not correlate with the typical clinical features of the disease. HSF1 inhibition reduced proopiomelanocortin (Pomc) transcription in AtT-20 cells. The HSF1 inhibitor KRIBB11 suppressed ACTH synthesis from 75% of human CD tumours in primary cell culture. This inhibitory action on Pomc transcription was mediated by increased glucocorticoid receptor and suppressed Nurr77/Nurr1 and AP-1 transcriptional activities. Conclusions: These data show that HSF1 regulates POMC transcription. Pharmacological targeting of HSF1 may be a promising treatment option for the control of excess ACTH secretion in CD.

FAM3C-YY1 axis is essential for TGFβ-promoted proliferation and migration of human breast cancer MDA-MB-231 cells via the activation of HSF1.

Family with sequence similarity three member C (FAM3C) (interleukin‐like EMT inducer [ILEI]), heat shock factor 1 (HSF1) and Ying‐Yang 1 (YY1) have been independently reported to be involved in the pathogenesis of various cancers. However, whether they are coordinated to trigger the development of cancer remains unknown. This study determined the role and mechanism of YY1 and HSF1 in FAM3C‐induced proliferation and migration of breast cancer cells. In human MDA‐MB‐231 breast cancer cell line, transforming growth factor‐β (TGFβ) up‐regulated FAM3C, HSF1 and YY1 expressions. FAM3C overexpression promoted the proliferation and migration of MDA‐MB‐231 cells with YY1 and HSF1 up‐regulation, whereas FAM3C silencing exerted the opposite effects. FAM3C inhibition repressed TGFβ‐induced HSF1 activation, and proliferation and migration of breast cancer cells. YY1 was shown to directly activate HSF1 transcription to promote the proliferation and migration of breast cancer cells. YY1 silencing blunted FAM3C‐ and TGFβ‐triggered activation of HSF1‐Akt‐Cyclin D1 pathway, and proliferation and migration of breast cancer cells. Inhibition of HSF1 blocked TGFβ‐, FAM3C‐ and YY1‐induced proliferation and migration of breast cancer cells. YY1 and HSF1 had little effect on FAM3C expression. Similarly, inhibition of HSF1 also blunted FAM3C‐ and TGFβ‐promoted proliferation and migration of human breast cancer BT‐549 cells. In human breast cancer tissues, FAM3C, YY1 and HSF1 protein expressions were increased. In conclusion, FAM3C activated YY1‐HSF1 signalling axis to promote the proliferation and migration of breast cancer cells. Furthermore, novel FAM3C‐YY1‐HSF1 pathway plays an important role in TGFβ‐triggered proliferation and migration of human breast cancer MDA‐MB‐231 cells.

RPPA-Profiling Identifies Patients with Low Phosphorylation Levels of HSF1 at Serine 326 As Potential Candidate for Bortezomib Treatment in Addition to Standard Therapy in Pediatric Acute Myeloid Leukemia

▪Background: Heat shock factor 1 (HSF1) is a key-component of the heat shock response and plays a major role in cancer biology. The heat shock response is a highly conserved mechanism that is important for cell survival under stressful conditions. It facilitates normal protein folding and guards the proteome from misfolding and aggregation. Bortezomib (BTZ) is a reversible proteasome inhibitor and was recently tested in a phase 3 clinical trial for patients with de novo pediatric acute myeloid leukemia (pedi-AML). This trial compared standard therapy (cytarabine/daunorubicin/etoposide (ADE)) to ADE plus BTZ (ADE+B). As HSF1 and BTZ act in opposing circumstances, our goal was to globally assess the phosphorylation of HSF1 at serine 326 (HSF1.pS326) and to determine if baseline HSF1.pS326 was prognostic of clinical response to ADE+B and if we could identify a subset of patients that benefitted from ADE+B.Methods: Reverse phase protein array (RPPA) probed with 222 total antibodies and 69 specific post-translationally modified proteins was used to determine the protein expression levels in 505 bulk leukemic pedi-AML samples compared to 20 CD34+ samples from healthy pediatric donors. Patients participated in the Children's Oncology Group (COG) AAML1031 Phase 3 clinical trial that compared ADE to ADE+B. Survival curves were generated using the Kaplan-Meier method.Results: HSF1.pS326 protein expression levels in the pedi-AML patient samples. Based on the HSF1.pS326 expression levels, patients were classified into two clusters; high HSF1 326 (n = 216) and low HSF1.326 (n = 289). The median of the normal CD34+ samples was used as cut-off point. Expression of HSF1.pS326 was not prognostic for outcome in patients treated with ADE (event-free survival (EFS), p = 0.51) (Fig. 1A). In patients that were treated with ADE+B however, HSF1.pS326 levels were highly prognostic, with low HSF1.pS326 associated with a better EFS (p = 0.009) (Fig. 1B). Patients with high HSF1.pS326 did not benefit from BTZ addition (EFS, p = 0.77) (Fig. 1C), whereas patients with low HSF1.pS326 levels significantly improved (EFS, p = 0.004) (Fig. 1D). Between the two patient clusters, the NPM1 mutation state was higher in patients with high HSF1.pS326 levels (p = 0.042), and the CEBPA mutation state was higher in patients with low HSF1.pS326 (p = 0.011). No other patient or disease characteristics were different between the two clusters.To functionally validate our findings, we overexpressed wild type or mutant HSF1 in 293T cells and assessed its effect on BTZ sensitivity. The mutant HSF1 constructs contained amino acid substitutions at Ser326, resulting in either a phospho-mimic HSF1 (S326E), or a phospho-dead HSF1 protein (S326A). Cell viability analysis showed clear resistance to BTZ in cells with increased phosphorylation (HSF1 S326E) (Fig. 1E - blue curve). Cells that expressed reduced phosphorylation of HSF1 (HSF1 S326A), however were highly sensitive to BTZ (Fig. 1E, red curve). This confirms our observations from the pedi-AML patients.Conclusions: Using a proteomics approach, we identified a subgroup of pedi-AML patients that benefitted from BTZ addition to ADE therapy. We hypothesize that patients with low HSF1 activation are more susceptible to protein cell stress, and protein cell stress susceptibility is amplified by the addition of BTZ with ADE. The use of HSF1.pS326 as potential biomarker could identify patients that would benefit from ADE+B therapy. Patients with high HSF1 could potentially benefit from ADE+B in combination with a HSF1 (phospho) inhibitor, suggesting a novel combinational therapy.Figure 1. Kaplan Meier survival curves for EFS based on HSF1.pS326 expression. A. HSF1.pS326 did not affect outcome in ADE treated patients (high HSF1.PS326 shown in red, low HSF1.pS326 in blue). B. HSF1.pS326 was highly prognostic for EFS ADE+B. C. Patients with high HSF1.pS326 did not benefit from BTZ (ADE shown in blue, ADE+B in green). D. Patients that expressed low HSF1.pS326 did significantly better after ADE+B. E. To assess the effect of high and low HSF1.pS326 on BTZ sensitivity, 293T cells were transfected with two HSF1.pS326 mutants that mimicked high (blue) and low (red) HSF1.pS326 patients, in addition to wild type HSF1 (orange) and an empty vector as control (green). Cell viability assays showed resistance to BTZ in the phospho-mimic (blue) mutant, whereas the phospho-dead (red) mutant was sensitive to BTZ. [Display omitted] DisclosuresKolb:Roche- Genentech: Membership on an entity's Board of Directors or advisory committees; Servier: Membership on an entity's Board of Directors or advisory committees.

Transcriptome profiling of Ricinus communis L. provides new insights underlying the mechanisms towards thermotolerance during seed imbibition and germination

Ricinus communis seeds germinate to a high percentage and faster at 35 °C than at lower temperatures, but with compromised seedling establishment and survival. However, seedlings are able to cope with high temperatures at later stages of seedling establishment if germination occurred at lower temperatures. The identification of this thermo-sensitive window during seed germination suggests that temperature disturb crucial mechanisms that support seedling establishment. We studied the molecular mechanisms that could explain this thermo-sensitive window with a genomics approach using microarray analysis to determine transcriptome changes during seed germination at 20, 25 and 35 °C. Although temperature had a strong effect on the R. communis transcriptome, most of these differences occurred between 6 h of imbibition and the commencement of germination, i.e. radicle protrusion, which coincided with the identified thermo-sensitive window. We identified several responsive genes that might be involved in the thermotolerance of R. communis. Temperature had a major effect on genes involved in energy generating pathways, such as the Calvin-Benson-Bassham cycle, gluconeogenesis, and starch- and triacylglycerol degradation. Transcripts of ATP binding proteins, DNA binding proteins, RNA binding proteins, DNA-directed RNA polymerases, heat shock factor proteins, multiprotein-bridging factor proteins, and zinc finger proteins were also affected by temperature suggesting that transcriptional reprogramming mechanisms were disturbed. Among the downregulated transcripts, three were shared by all three stages: one oxidation-related zinc finger 2, one F-box and wd40 domain protein, and one DNA binding protein/ MYB-like transcription factor. Among the upregulated transcripts, nine were shared by all three stages: one BET1P/SFT1P-like protein 14BB, one low-molecular-weight cysteine-rich protein LCR78, one WD-repeat protein, one GAST1 protein, one adenylate kinase 1/P-loop containing nucleoside triphosphate hydrolases superfamily protein, and four conserved hypothetical proteins. These genes constitute good candidate genes for further characterization of temperature-responsive molecular mechanisms in R. communis, which in turn will provide necessary tools for the exploitation of R. communis by small family farmers under the typical harsh conditions of semiarid regions worldwide.

Genetic polymorphism and expression of HSF1 gene is significantly associated with breast cancer in Saudi females.

The transcription factor, heat shock factor 1 (HSF1), influences the expression of heat shock proteins as well as other activities like the induction of tumor suppressor genes, signal transduction pathway, and glucose metabolism. We hypothesized that single nucleotide polymorphisms (SNPs) in HSF1 gene might affect its expression or function which might have an influence on the development of breast cancer. The study group included 242 individuals (146 breast cancer patients and 96 healthy controls). From the cancer patients, genomic DNA was extracted from 96 blood samples and 50 Formalin-Fixed Paraffin Embedded (FFPE) tissues, while from the controls DNA were extracted from blood only. Genotype was carried out for four SNPs in the HSF1 gene (rs78202224, rs35253356, rs4977219 and rs34404564) using Taqman genotyping assay method. The HSF1 expression was investigated using immunohistochemistry on FFPE tissues (cancer tissue and adjacent normal tissue). The SNP rs78202224 (G>T) was significantly associated with increased risk of breast cancer. The combined TT + GT genotype (OR: 6.91; p: 0.035) and the T allele showed high risk (OR: 5.81; p:0.0085) for breast cancer development. The SNP rs34404564 (A>G) had a protective effect against the development of breast cancer. The genotype AG (OR: 0.41; p = 0.0059) and GG+AG (OR: 0.52; p: 0.026) occurred at a significantly lower frequency in the breast cancer patients compared to the frequency in healthy controls. No significant relationship was identified between either rs35253356 (A>G) or rs4977219 (A>C) and breast cancer in Saudi. The HSF1 protein expression was higher in all invasive and in situ breast carcinoma compared to the normal tissue. A stronger positive staining for HSF1 was found in the nucleus compared to the cytoplasm. Our results show that HSF1 gene expression is elevated in breast cancer tissue and two of the studied SNPs correlate significantly with cancer development.

Abstract 4459: Glycine decarboxylase inhibits proliferation of ovarian cancer cells via SIRT1-mediated apoptosis

Abstract Glycine decarboxylase (GLDC) is a very recently identified metabolic oncogene that links glycine metabolism with tumorigenesis. We designed this study to determine the mechanistic role of GLDC in in ovarian cancer cells. First, we confirmed the expressions of GLDC in eight different ovarian cancer cell lines. And colony formation assay revealed decreased proliferation of OVCAR3 ovarian cancer cells transfected with siGLDC compared with that of control. Intriguingly, we found that knockdown of GLDC induced decreased S-adenosylmethionine (SAM)/ S-adenosylhomocysteine (SAH) ratio, which implicates methyl donor deficient state in OVCAR3 cells. Expressions of sirtuin 1 (SIRT1), heat shock protein (HSP) 90 and heat-shock factor protein 1 (HSF1) decreased while that of p53 increased in GLDC knock-downed cells, suggesting heat shock response. Addition of SAM into siGLDC knock-downed cell restored the expression of SIRT1. Inhibition of SIRT1 decreased Bcl2 and increased cytochrome c expressions. In summary, these results suggest GLDC induces methyl donor deficient state in OVCAR3 ovarian cancer cells, which in turn leads to apoptosis via SIRT1-mediated pathway. Citation Format: So-Jin Shin, Jin-Young Kim, Eun-Young Ha, Chi-Heum Cho. Glycine decarboxylase inhibits proliferation of ovarian cancer cells via SIRT1-mediated apoptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4459. doi:10.1158/1538-7445.AM2017-4459

HSF1 acetylation decreases its transcriptional activity and enhances glucolipotoxicity-induced apoptosis in rat and human beta cells.

Heat shock factor protein 1 (HSF1) is a transcription factor that regulates the expression of key molecular chaperones, thereby orchestrating the cellular response to stress. This system was recently implicated in the control of insulin sensitivity and is therefore being scrutinised as a novel therapeutic avenue for type 2 diabetes. However, the regulation and biological actions of HSF1 in beta cells remain elusive. Herein, we sought to investigate the regulation of HSF1 in pancreatic beta cells and to study its potential role in cell survival. We exposed human islets and beta cell lines to glucolipotoxicity and thapsigargin. HSF1 activity was evaluated by gel shift assay. HSF1 acetylation and interaction with the protein acetylase cAMP response element binding protein (CBP) were investigated by western blot. We measured the expression of HSF1 and its canonical targets in islets from Goto-Kakizaki (GK) rat models of diabetes and delineated the effects of HSF1 acetylation using mutants mimicking constitutive acetylation and deacetylation of the protein. Glucolipotoxicity promoted HSF1 acetylation and interaction with CBP. Glucolipotoxicity-induced HSF1 acetylation inhibited HSF1 DNA binding activity and decreased the expression of its target genes. Restoration of HSF1 activity in beta cells prevented glucolipotoxicity-induced endoplasmic reticulum stress and apoptosis. However, overexpression of a mutant protein (K80Q) mimicking constitutive acetylation of HSF1 failed to confer protection against glucolipotoxicity. Finally, we showed that expression of HSF1 and its target genes were altered in islets from diabetic GK rats, suggesting that this pathway could participate in the pathophysiology of diabetes and constitutes a potential site for therapeutic intervention. Our results unravel a new mechanism by which HSF1 inhibition is required for glucolipotoxicity-induced beta cell apoptosis. Restoring HSF1 activity may represent a novel strategy for the maintenance of a functional beta cell mass. Our study supports the therapeutic potential of HSF1/heat shock protein-targeting agents in diabetes treatment.

Unexpected heat shock element binding ability and tumor-killing activity of the combinatorial function domain of apoptin.

Apoptin, derived from the chicken anemia virus, has been found to exert tumor-preferential apoptotic activity. It is a potential anticancer agent with direct clinical applications. However, if this viral protein were to be used as a new drug, it might also induce a strong immune response, causing toxic side effects. In a previous study, our group showed that TAT-apoptin downregulates the stress expression of heat shock protein 70 by competing with heat shock factor protein 1 in binding to the heat shock element (HSE) of the promoter region of heat shock protein 70, thus inducing specific apoptosis in HepG2 cells. In this study, we investigated the HSE-binding properties of the minimal functional region of apoptin. We showed that apoptin's nuclear localization signals 1 and nuclear localization signals 2 represented functional regions that could bind with HSE and that this binding capacity was increased by polymers formed through the introduction of a leucine-rich stretch. Our data also showed that truncated combinatorial apoptin peptide has greater tumor-specific cell-killing activity and could be a potential antitumor agent.

The prognostic utility of the transcription factor SRF in docetaxel-resistant prostate cancer: in-vitro discovery and in-vivo validation

BackgroundDocetaxel based therapy is one of the first line chemotherapeutic agents for the treatment of metastatic castrate-resistant prostate cancer. However, one of the major obstacles in the treatment of these patients is docetaxel-resistance. Defining the mechanisms of resistance so as to inform subsequent treatment options and combinations represents a challenge for clinicians and scientists.Previous work by our group has shown complex changes in pro and anti-apoptotic proteins in the development of resistance to docetaxel. Targeting these changes individually does not significantly impact on the resistant phenotype but understanding the central signalling pathways and transcription factors (TFs) which control these could represent a more appropriate therapeutic targeting approach.MethodsUsing a number of docetaxel-resistant sublines of PC-3 cells, we have undertaken a transcriptomic analysis by expression microarray using the Affymetrix Human Gene 1.0 ST Array and in conjunction with bioinformatic analyses undertook to predict dysregulated TFs in docetaxel resistant prostate cancer. The clinical significance of this prediction was ascertained by performing immunohistochemical (IHC) analysis of an identified TF (SRF) in the metastatic sites from men who died of advanced CRPC. Investigation of the functional role of SRF was examined by manipulating SRF using SiRNA in a docetaxel-resistant PC-3 cell line model.ResultsThe transcription factors identified include serum response factor (SRF), nuclear factor kappa-B (NFκB), heat shock factor protein 1 (HSF1), testicular receptor 2 & 4 (TR2 &4), vitamin-D and retinoid x receptor (VDR-RXR) and oestrogen-receptor 1 (ESR1), which are predicted to be responsible for the differential gene expression observed in docetaxel-resistance. IHC analysis to quantify nuclear expression of the identified TF SRF correlates with both survival from date of bone metastasis (p = 0.003), survival from androgen independence (p = 0.00002), and overall survival from prostate cancer (p = 0.0044). Functional knockdown of SRF by siRNA demonstrated a reversal of apoptotic resistance to docetaxel treatment in the docetaxel-resistant PC-3 cell line model.ConclusionsOur results suggest that SRF could aid in treatment stratification of prostate cancer, and may also represent a therapeutic target in the treatment of men afflicted with advanced prostate cancer.

Heat shock 70 kDa protein cognate 5 involved in WSSV toleration of Litopenaeus vannamei

The expression levels of 97 unigenes encoding heat shock proteins of Litopenaeus vannamei was scanned, and ten of them were significantly induced by white spot syndrome virus (WSSV). Among these genes, heat shock 70 kDa protein cognate 5 (LvHSC70-5) was upregulated to the highest extent and subjected to further studies. Subcellular localization assay revealed that LvHSC70-5 was located in the mitochondria. Aside from WSSV infection, unfolded protein response activation and thermal stress could also upregulate LvHSC70-5. Results of reporter gene assay demonstrated that promoter of LvHSC70-5 was activated by L. vannamei heat shock factor protein 1, activating transcription factor 4 and thermal stress. A decrease in the expression of LvHSC70-5 could reduce the aggregation of proteins in hemocytes and the cumulative mortality of WSSV-infected L. vannamei. LvHSC70-5 in L. vannamei hemocytes was upregulated by mild thermal stress. In addition, mild thermal stress, decreased the copy number of WSSV in shrimp muscle and the cumulative mortality of WSSV-infected L. vannamei. Therefore, collecting results suggested that LvHSC70-5 should be involved in WSSV toleration of shrimp L. vannamei.

Protection of Glial Müller Cells by Dexamethasone in a Mouse Model of Surgically Induced Blood-Retinal Barrier Breakdown.

Breakdown of the inner blood-retinal barrier (iBRB) occurs in many retinal disorders and may cause retinal edema often responsible for vision loss. Dexamethasone is used in clinical practice to restore iBRB. The aim of this study was to characterize the impact of a surgically induced iBRB breakdown on retinal homeostatic changes due to dystrophin Dp71, aquaporin-4 (AQP4), and Kir4.1 alterations in Müller glial cells (MGC) in a mouse model. The protective effect of dexamethasone was assessed in this model. Moreover, retinal explants were used to control MGC exposure to a hypoosmotic solution containing barium. Partial lens surgery was performed in C57BL6/J mice. Dystrophin Dp71, AQP4, and Kir4.1 expression was analyzed by quantitative RT-PCR, Western blot, and immunohistochemistry. Twenty-four hours after surgery, mice received a single intravitreal injection of dexamethasone or of vehicle. After partial lens surgery, iBRB permeability increased while Dp71 and AQP4 were downregulated and Kir4.1 was delocalized. These effects were partially prevented by dexamethasone injection. In the retinal explant model, MGC were swollen and Dp71, AQP4, and Kir4.1 were downregulated after exposure to a hypoosmotic solution containing barium, but not in the presence of dexamethasone. Heat shock factor protein 1 (HSF1) was overexpressed in dexamethasone-treated retinas. Partial lens surgery induces iBRB breakdown and molecular changes in MGC, including a downregulation of Dp71 and AQP4 and the delocalization of Kir4.1. Dexamethasone seems to protect retina from these molecular changes by upregulating HSF1.

Overexpression of heat-shock factor 1 is associated with a poor prognosis in esophageal squamous cell carcinoma.

Heat-shock factor 1 (HSF1) is the primary regulator of the response to various stressors. A previous study showed that HSF1 expression is associated with a poor prognosis in breast cancer and hepatocellular carcinoma; however, the prognostic significance of HSF1 in esophageal squamous cell carcinoma (ESCC) is unknown. Therefore, the present study investigated the association between HSF1 expression and the clinicopathological parameters of patients, as well as the association between HSF1 expression, and heat shock protein (Hsp)27, Hsp70 and Hsp90 expression induced by HSF1, by cDNA microarray and immunohistochemistry analyses. HSF1 protein and mRNA expression were assessed in resected specimens from 270 patients with ESCC in two independent cohorts. Hsp27, Hsp70 and Hsp90 expression were also assessed in 55/270 patients. Patients with high HSF1 expression had a significantly worse OS than those with low HSF1 expression in both cohorts. In multivariate analyses, pathological T stage [hazard ratio (HR), 2.21; 95% confidence interval (CI), 1.38-3.65; P=0.0008], pathological N stage (HR, 1.73; 95% CI, 1.04-3.02; P=0.03) and HSF1 expression (HR, 2.29; 95% CI, 1.48-3.64; P=0.0002) were statistically significant independent prognostic factors. Furthermore, Hsp27 and Hsp90 expression were significantly correlated with HSF1 expression (P<0.0001), but Hsp70 expression was not (P=0.38). These results indicate that HSF1 is a prognostic factor for patients with ESCC, and that Hsp27 and Hsp90, but not Hsp70, may be the downstream targets of HSF1 in ESCC.

Control of Secreted Protein Gene Expression and the Mammalian Secretome by the Metabolic Regulator PGC-1α

Secreted proteins serve pivotal roles in the development of multicellular organisms, acting as structural matrix, extracellular enzymes, and signal molecules. However, how the secretome is regulated remains incompletely understood. Here we demonstrate, unexpectedly, that peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α), a critical transcriptional co-activator of metabolic gene expression, functions to down-regulate the expression of diverse genes encoding secreted molecules and extracellular matrix components to modulate the secretome. Using cell lines, primary cells, and mice, we show that both endogenous and exogenous PGC-1α down-regulate the expression of numerous genes encoding secreted molecules. Mechanistically, results obtained using mRNA stability measurements as well as intronic RNA expression analysis are consistent with a transcriptional effect of PGC-1α on the expression of genes encoding secreted proteins. Interestingly, PGC-1α requires the central heat shock response regulator heat shock factor protein 1 (HSF1) to affect some of its targets, and both factors co-reside on several target genes encoding secreted molecules in cells. Finally, using a mass spectrometric analysis of secreted proteins, we demonstrate that PGC-1α modulates the secretome of mouse embryonic fibroblasts. Our results define a link between a key pathway controlling metabolic regulation and the regulation of the mammalian secretome.

HSP70-HSF1 Interplays Has a Role in the Pathogenesis of Chronic Lymphocytic Leukemia and Is a Druggable Target

INTRODUCTIONWe recently found that the Heat Shock Protein of 70kDa (HSP70), an ATP-dependent chaperone that is induced by cellular stress and protects cells against various apoptotic stimuli, was particularly overexpressed in neoplastic B cells from Chronic Lymphocytic Leukemia (CLL) vs normal B lymphocytes. HSP70 responds to a wide variety of physiological and environmental stress signals, thus allowing cells to survive to lethal conditions. The primary responsible for the transcription of HSP70 is the heat shock factor 1 (HSF1), being the major regulator of HSP70 expression. In response to stress, HSF1 becomes phosphorylated, forms homotrimers, binds DNA and activates heat shock gene transcription.Considering that the search for molecules involved in the apoptosis resistance and increased survival of B cells from CLL is still ongoing, with this as a background, we were aimed at studying and targeting HSP70 or players related to it (i.e. HSF1) in view of their clinical, prognostic and therapeutical relevance in CLL.METHODSHSP70/HSF1 axis was analysed in freshly isolated leukemic B cells from CLL patients. Expression levels of HSP70, HSF1 and HSF1-Ser326 were assessed by Western blotting analysis with specific antibodies and the obtained expression data have been correlated with clinical features of the patients. HSP70 subcellular localization has been determined by confocal microscopy and cell fractionation. HSP70 expression and localization was also assessed by immunohistochemistry in lymph nodes from CLL patients. Leukemic B cells from 15 CLL therapy-free patients were treated with different concentrations of: i) Zafirlukast, an oral leukotriene receptor antagonist used to prevent asthma symptoms and acting also as HSP70 inhibitor and ii) Fisetin, a dietary flavonoid acting as anti-inflammatory and anti-carcinogen, that inhibits HSF1 activity through the block of its binding to the HSP70 promoter. Apoptosis induction in CLL cells was evaluated by Annexin V/Propidium Iodide flow cytometry test and by the presence of cleaved PARP observed in Western blotting.RESULTSWe found that HSP70 and HSF1 proteins were overexpressed in leukemic vs normal B cells and correlated to poor prognosis. In particular, IGHV unmutated or ZAP70 positive patients presented higher levels of HSP70 and HSF1 with respect to patients with a favorable prognosis. Moreover, the two proteins presented a positive correlation (p<0.0001, r=0.84; Pearson’s correlation) thus hypothesizing a positive loop feedback for their expression. We found that, in CLL, HSF1 was constitutively phosphorylated at activatory Ser326, thus being positively regulated, in a large part of our patients. In addition, patients presenting a higher phosphorylation of HSF1 at Ser326 were mostly ZAP70 positive patients. We also observed an abnormal constitutive nuclear localization of HSP70 in leukemic cells. On the basis of these results and the pro-survival role played by HSP70 and HSF1, we analyzed the effects of their inhibition in leukemic cells of our patients by using two inhibitors of this axis, Zafirlukast and Fisetin. Both inhibitors have been proven to be effective in inducing a dose-dependent cell apoptosis in CLL B cells.CONCLUSIONSHSP70 overexpression is involved in a diminished response to treatment by promoting the adaptation of tumor cells to changes (i.e. toxic conditions) currently induced by chemotherapy thus revealing critical roles for HSP70 in cancer initiation and progression. It has been shown that HSP70 depletion results in an increased sensitivity to chemotherapy. For this reason, and considering its prognostic implications and functional role in cancers, including CLL, HSP70 represents an interesting target for antileukemic therapies.In this context, our results suggest: i) an involvement of HSP70/HSF1 axis in the pathogenesis of CLL; ii) an input for further studies that consider the possible involvement of ZAP70 in HSP70/HSF1 axis in CLL; iii) the putative usage of Zafirlukast, which is a drug already available for clinical use and in the targeting of HSF1 in CLL. DisclosuresNo relevant conflicts of interest to declare.