ER Stress-Activated HSF1 Governs Cancer Cell Resistance to USP7 Inhibitor-Based Chemotherapy through the PERK Pathway.

Ubiquitin-specific protease 7 inhibitors (USP7i) are considered a novel class of anticancer drugs. Cancer cells occasionally become insensitive to anticancer drugs, known as chemoresistance, by acquiring multidrug resistance, resulting in poor clinical outcomes in patients with cancer. However, the chemoresistance of cancer cells to USP7i (P22077 and P5091) and mechanisms to overcome it have not yet been investigated. In the present study, we generated human cancer cells with acquired resistance to USP7i-induced cell death. Gene expression profiling showed that heat stress response (HSR)- and unfolded protein response (UPR)-related genes were largely upregulated in USP7i-resistant cancer cells. Biochemical studies showed that USP7i induced the phosphorylation and activation of heat shock transcription factor 1 (HSF1), mediated by the endoplasmic reticulum (ER) stress protein kinase R-like ER kinase (PERK) signaling pathway. Inhibition of HSF1 and PERK significantly sensitized cancer cells to USP7i-induced cytotoxicity. Our study demonstrated that the ER stress-PERK axis is responsible for chemoresistance to USP7i, and inhibiting PERK is a potential strategy for improving the anticancer efficacy of USP7i.

Heat shock transcription factor 1 is SUMOylated in the activated trimeric state.

The heat shock response is a transcriptional program of organisms to counteract an imbalance in protein homeostasis. It is orchestrated in all eukaryotic cells by heat shock transcription factor 1 (Hsf1). Despite very intensive research, the intricacies of the Hsf1 activation-attenuation cycle remain elusive at a molecular level. Post-translational modifications belong to one of the key mechanisms proposed to adapt the Hsf1 activity to the needs of individual cells, and phosphorylation of Hsf1 at multiple sites has attracted much attention. According to cell biological and proteomics data, Hsf1 is also modified by small ubiquitin-like modifier (SUMO) at several sites. How SUMOylation affects Hsf1 activity at a molecular level is still unclear. Here, we analyzed Hsf1 SUMOylation in vitro with purified components to address questions that could not be answered in cell culture models. In vitro Hsf1 is primarily conjugated at lysine 298 with a single SUMO, though we did detect low-level SUMOylation at other sites. Different SUMO E3 ligases such as protein inhibitor of activated STAT 4 enhanced the efficiency of in vitro modification but did not alter SUMO site preferences. We provide evidence that Hsf1 trimerization and phosphorylation at serines 303 and 307 increases SUMOylation efficiency, suggesting that Hsf1 is SUMOylated in its activated state. Hsf1 can be SUMOylated when DNA bound, and SUMOylation of Hsf1 does neither alter DNA-binding affinity nor affects heat shock cognate 71kDa protein (HSPA8)+DnaJ homolog subfamily B member 1-mediated monomerization of Hsf1 trimers and concomitant dislocation from DNA. We propose that SUMOylation acts at the transcription level of the heat shock response.

Comparative interactomes of HSF1 in stress and disease reveal a role for CTCF in HSF1-mediated gene regulation.

Heat shock transcription factor 1 (HSF1) orchestrates cellular stress protection by activating or repressing gene transcription in response to protein misfolding, oncogenic cell proliferation, and other environmental stresses. HSF1 is tightly regulated via intramolecular repressive interactions, post-translational modifications, and protein-protein interactions. How these HSF1 regulatory protein interactions are altered in response to acute and chronic stress is largely unknown. To elucidate the profile of HSF1 protein interactions under normal growth and chronic and acutely stressful conditions, quantitative proteomics studies identified interacting proteins in the response to heat shock or in the presence of a poly-glutamine aggregation protein cell-based model of Huntington's disease. These studies identified distinct protein interaction partners of HSF1 as well as changes in the magnitude of shared interactions as a function of each stressful condition. Several novel HSF1-interacting proteins were identified that encompass a wide variety of cellular functions, including roles in DNA repair, mRNA processing, and regulation of RNA polymerase II. One HSF1 partner, CTCF, interacted with HSF1 in a stress-inducible manner and functions in repression of specific HSF1 target genes. Understanding how HSF1 regulates gene repression is a crucial question, given the dysregulation of HSF1 target genes in both cancer and neurodegeneration. These studies expand our understanding of HSF1-mediated gene repression and provide key insights into HSF1 regulation via protein-protein interactions.

Aspirin relieves the calcification of aortic smooth muscle cells by enhancing the heat shock response.

CONTEXT: Vascular calcification is a major complication of chronic renal failure, which has been identified as an active process partly driven by osteogenic transition of vascular smooth muscle cells (VSMCs). Aspirin could prevent cardiomyocyte damage by inducing heat shock response. OBJECTIVE: This study investigates the effect of aspirin on alleviating VSMC calcification. MATERIALS AND METHODS: An in vitro VSMC calcification model was established by 10-day calcification induction in osteogenic medium. VSMCs were grouped as following: control group (normal medium), calcified group (osteogenic medium) and treated group (osteogenic medium with 1 or 4 mmol/L aspirin). VSMC calcification was evaluated by calcified nodules formation, intracellular calcium concentration and osteoblastic marker (OPN and Runx2) expression. RESULTS: After 10-day culture, the intracellular calcium concentration in calcified group was significantly higher than that in control group (1.16 ± 0.04 vs. 0.14 ± 0.01 µg/mg, p < 0.01), but significantly reduced in 1 mmol/L aspirin treated group (0.74 ± 0.05 µg/mg, p < 0.01), and 4 mmol/L aspirin treated group (0.93 ± 0.03 µg/mg, p < 0.01). The elevated expression of OPN and Runx2 induced by osteogenic medium was significantly relieved after 1 or 4 mmol/L aspirin treatment. The expression of HSF1, HSP70 and HSP90 was decreased in calcification-induced VSMCs, but significantly increased after treatment of aspirin. Furthermore, inhibition of HSP70 (or HSP90) by small-molecule inhibitor or small interfering RNA could partially abolish the anti-calcification effect of aspirin, proved by the changes of intracellular calcium concentration and osteoblastic marker expression. DISCUSSION AND CONCLUSIONS: Aspirin could relieve the calcification of VSMCs partially through HSP70- or HSP90-mediated heat shock response. These findings expanded the understanding of aspirin pharmacology, and imply that local induction expression of HSPs might be a potential therapeutic strategy for the prevention and therapy of vascular calcification.

[Overexpressed HSF1 and 4-HNE relate to the malignant phenotype of prostate cancer].

OBJECTIVE: To investigate the expressions of HSF1 and tetra-hydroxynonene (4-HNE) in prostate cancer (PCa) tissue and their clinical significance. METHODS: Using the immunohistochemical method, we detected the expressions of HSF1 and 4-HNE in the prostatic tissues of 50 cases of PCa and another 50 cases of BPH, and analyzed the relationship of the two expressions with Gleason grade. RESULTS: The positive expression level of HSF1 was dramatically higher in the highly, moderately and lowly differentiated PCa than in the BPH tissue (37.5%, 50.0% and 75.0% vs 4.0%, P = 0.001) and showed statistically significant difference among different Gleason grades (P = 0.025), so was that of 4-HNE (81.3%, 92.9% and 100.0% % vs 6.0%, P = 0.001), also with statistically significant difference among different Gleason grades (P = 0.029). There was a positive correlation between the expression of HSF1 and that of 4-HNE in the PCa tissue (r = 0.947, P = 0.001). CONCLUSIONS: The overexpressions of HSF1 and 4-HNE are related to the Gleason grades of prostate cancer, which can be used as an new biological marker with important reference value for assessing the malignancy of prostate cancer.

Protective role of heat shock transcription factor 1 in heart failure: A diagnostic approach.

OBJECTIVE: Nonexpression or expression inhibition of protective factors has been determined in the occurrence of heart failure (HF). Heat shock transcription factor 1 (HSF1) is among such factors, which reduces the incidence of HF by controlling cardiac hypertrophy and fibrosis. In this study, molecular mechanisms for nonexpression of HSF1 in HF patients have been investigated. MATERIALS AND METHODS: This review paper is based on the material obtained via PubMed search of 1996-2018. The key search terms were "heart failure," "heat shock transcription factor 1," "hypertrophy", "fibrosis," and "apoptosis." RESULTS: Although factors such as janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) and heat shock proteins (HSPs) may respectively increase and decrease susceptibility to HF, in some circumstances, these factors may unexpectedly prevent HF progression. CONCLUSION: Finally, identification of molecular pathways expressed by various factors could be used to design appropriate treatments or to employ strategies inducing the expression of HSF1 to prevent HF.

Heat-shock transcription factor 1 is critically involved in the ischaemia-induced cardiac hypertrophy via JAK2/STAT3 pathway.

Cardiac hypertrophy after myocardial infarction (MI) is an independent risk factor for heart failure. Regression of cardiac hypertrophy has emerged as a promising strategy in the treatment of MI patients. Here, we have been suggested that heat-shock transcription factor 1 (HSF1) is a novel repressor of ischaemia-induced cardiac hypertrophy. Ligation of left anterior descending coronary was used to produce MI in HSF1-deficient heterozygote (KO), HSF1 transgenic (TG) mice and their wild-type (WT) littermates, respectively. Neonatal rat cardiomyocytes (NRCMs) were treated by hypoxia to mimic MI in vitro. The HSF1 phosphorylation was significantly reduced in the infarct border zone of mouse left ventricles (LVs) 1 week after MI and in the hypoxia-treated NRCMs. HSF1 KO mice showed more significant maladaptive cardiac hypertrophy and deteriorated cardiac dysfunction 1 week after MI compared to WT MI mice. Deficiency of HSF1 by siRNA transfection notably increased the hypoxia-induced myocardial hypertrophy in NRCMs. Mechanistically, Janus kinase 2 (JAK2) and its effector, signal transducer and activator of transcription 3 (STAT3) were found to be significantly increased in the LV infarct border zone of WT mice after MI as well as the NRCMs treated by hypoxia. These alterations were more significant in HSF1 KO mice and NRCMs transfected with HSF1 SiRNA. Inversely, HSF1 TG mice showed significantly ameliorated cardiac hypertrophy and heart failure 1 week after LAD ligation compared to their WT littermates. Our data collectively demonstrated that HSF1 is critically involved in the pathological cardiac hypertrophy after MI via modulating JAK2/STAT3 signalling and may constitute a potential therapeutic target for MI patients.

Homocysteine facilitates LOX-1 activation and endothelial death through the PKCß and SIRT1/HSF1 mechanism: relevance to human hyperhomocysteinaemia.

HHcy (hyperhomocysteinaemia) is one of the major risk factors for cardiovascular diseases. A high concentration of Hcy (homocysteine) induces endothelial dysfunction by activating endothelial oxidative stress. LOX-1 (lectin-like oxidized low-density lipoprotein receptor 1) plays a vital role in regulating the progression of atherosclerotic lesions. LOX-1 activation causes endothelial apoptosis and inflammation. The mechanism is still unclear as to whether Hcy affects human endothelial LOX-1 expression. LOX-1 expression level was confirmed by Western blotting assay in Hcy-treated endothelial cells. L-Methionine was used for HHcy induction in animals. Our results suggested that Hcy increased PKCß (protein kinase Cß) activation to enhance the LOX-1 expression level. The up-regulation of PKCß phosphorylation subsequently causes ROS (reactive oxygen species) formation and SIRT1 (sirtuin 1) degradation through a proteasome-dependent mechanism, thereby mitigating the activity of SIRT1 by deacetylating HSF1 (heat-shock transcription factor 1). We also found that NOX2 is a key NAPDH oxidase isoform responsible for the Hcy-caused ROS formation. The overexpression of SIRT1 and HSF1 reduced the Hcy-induced LOX-1 activation. Silencing PKCß function also reduced LOX-1 activation and endothelial apoptosis caused by Hcy. Our hypothesis was supported by analysing the data from methionine-induced HHcy-affected animals. Our data indicate a new direction for LOX-1 regulation by the modulation of the PKCß/NAPDH oxidase/SIRT1/HSF1 mechanism. Our findings might provide a novel route for developing new therapeutic treatments for HHcy.

Regulation of autophagy by stress-responsive transcription factors.

Autophagy is an evolutionarily conserved process that promotes the lysosomal degradation of intracellular components including organelles and portions of the cytoplasm. Besides operating as a quality control mechanism in steady-state conditions, autophagy is upregulated in response to a variety of homeostatic perturbations. In this setting, autophagy mediates prominent cytoprotective effects as it sustains energetic homeostasis and contributes to the removal of cytotoxic stimuli, thus orchestrating a cell-wide, multipronged adaptive response to stress. In line with the critical role of autophagy in health and disease, defects in the autophagic machinery as well as in autophagy-regulatory signaling pathways have been associated with multiple human pathologies, including neurodegenerative disorders, autoimmune conditions and cancer. Accumulating evidence indicates that the autophagic response to stress may proceed in two phases. Thus, a rapid increase in the autophagic flux, which occurs within minutes or hours of exposure to stressful conditions and is entirely mediated by post-translational protein modifications, is generally followed by a delayed and protracted autophagic response that relies on the activation of specific transcriptional programs. Stress-responsive transcription factors including p53, NF-κB and STAT3 have recently been shown to play a major role in the regulation of both these phases of the autophagic response. Here, we will discuss the molecular mechanisms whereby autophagy is orchestrated by stress-responsive transcription factors.

AICAR-induced activation of AMPK negatively regulates myotube hypertrophy through the HSP72-mediated pathway in C2C12 skeletal muscle cells.

5'-AMP-activated protein kinase (AMPK) plays an important role as a negative regulator of skeletal muscle mass. However, the precise mechanism of AMPK-mediated regulation of muscle mass is not fully clarified. Heat shock proteins (HSPs), stress-induced molecular chaperones, are related with skeletal muscle adaptation, but the association between AMPK and HSPs in skeletal muscle hypertrophy is unknown. Thus, we investigated whether AMPK regulates hypertrophy by mediating HSPs in C2C12 cells. The treatment with AICAR, a potent stimulator of AMPK, decreased 72-kDa HSP (HSP72) expression, whereas there were no changes in the expressions of 25-kDa HSP, 70-kDa heat shock cognate, and heat shock transcription factor 1 in myotubes. Protein content and diameter were less in the AICAR-treated myotubes in those without treatment. AICAR-induced suppression of myotube hypertrophy and HSP72 expression was attenuated in the siRNA-mediated AMPKα knockdown myotubes. AICAR increased microRNA (miR)-1, a modulator of HSP72, and the increase of miR-1 was not induced in AMPKα knockdown condition. Furthermore, siRNA-mediated HSP72 knockdown blocked AICAR-induced inhibition of myotube hypertrophy. AICAR upregulated the gene expression of muscle Ring-finger 1, and this alteration was suppressed in either AMPKα or HSP72 knockdown myotubes. The phosphorylation of p70 S6 kinase Thr(389) was downregulated by AICAR, whereas this was attenuated in AMPKα, but not in HSP72, knockdown myotubes. These results suggest that AMPK inhibits hypertrophy through, in part, an HSP72-associated mechanism via miR-1 and protein degradation pathways in skeletal muscle cells.

Changes in cardiac thrombomodulin and heat shock transcription factor 1 expression and peripheral thrombomodulin and catecholamines during hypothermia in rats.

Effects of hypothermia and rewarming on thrombomodulin, catecholamines and heat shock transcription factor 1 (HSF1) were studied in rats. The aims of this study were to clarify whether cold stress, under anesthesia, is sufficient to change levels of thrombomodulin in healthy endothelium and in the circulation and whether adrenaline, noradrenaline and HSF1 could act as regulators in the process. Rats were divided into control, mild hypothermia (2 and 4.5 hours at + 21 °C; MH1, MH2), severe hypothermia (2 and 4.5 h at + 10 °C; SH1, SH2) and two rewarming groups (2 h at + 10 °C followed by 2 h at + 21 °C or 3 h at + 28 °C; SHW1, SHW2) (n = 15/group, except n = 6 in MH1). Fentanyl-fluanisone-midazolam was used as anesthetic. Low levels of thrombomodulin in plasma and myocardial arterioles/venules measured by ELISA and immunohistochemistry were associated with significant increase of thrombomodulin transcript level in SH1 rats analyzed by quantitative PCR. Plasma adrenaline correlated negatively with the relative amount of myocardial thrombomodulin transcripts and positively with plasma thrombomodulin in SH. Transcript levels of thrombomodulin and HSF1 correlated strongly (r = 0.83; p < 0.001) in SH. Plasma/urine ratio of thrombomodulin and plasma adrenaline (r = 0.87; p = 0.005) or noradrenaline (r = 0.78; p = 0.023) were strongly correlated in SHW1 rats. Hence, cellular and soluble levels of thrombomodulin are modified by cold stress in healthy rats, possibly via catecholamines and HSF1.

BAP1 regulates HSF1 activity and cancer immunity in pancreatic cancer.

BACKGROUND: The vast majority of pancreatic cancers have been shown to be insensitive to single-agent immunotherapy. Exploring the mechanisms of immune resistance and implementing combination therapeutic strategies are crucial for PDAC patients to derive benefits from immunotherapy. Deletion of BAP1 occurs in approximately 27% of PDAC patients and is significantly correlated with poor prognosis, but the mechanism how BAP1-deletion compromises survival of patients with PDAC remain a puzzle. METHODS: Bap1 knock-out KPC (KrasG12D/+; LSLTrp53R172H/+; Pdx-1-Cre) mice and control KPC mice, syngeneic xenograft models were applied to analysis the correlation between BAP1 and immune therapy response in PDAC. Immunoprecipitation, RT-qPCR, luciferase and transcriptome analysis were combined to revealing potential mechanisms. Syngeneic xenograft models and flow cytometry were constructed to examine the efficacy of the inhibitor of SIRT1 and its synergistic effect with anti-PD-1 therapy. RESULT: The deletion of BAP1 contributes to the resistance to immunotherapy in PDAC, which is attributable to BAP1's suppression of the transcriptional activity of HSF1. Specifically, BAP1 competes with SIRT1 for binding to the K80 acetylated HSF1. The BAP1-HSF1 interaction preserves the acetylation of HSF1-K80 and promotes HSF1-HSP70 interaction, facilitating HSF1 oligomerization and detachment from the chromatin. Furthermore, we demonstrate that the targeted inhibition of SIRT1 reverses the immune insensitivity in BAP1 deficient PDAC mouse model. CONCLUSION: Our study elucidates an unrevealed mechanism by which BAP1 regulates immune therapy response in PDAC via HSF1 inhibition, and providing promising therapeutic strategies to address immune insensitivity in BAP1-deficient PDAC.

Astragaloside IV ameliorates pressure overload-induced heart failure by enhancing angiogenesis through HSF1/VEGF pathway.

Astragaloside IV(AS-IV), the main active ingredient of Astragalus, has been used as a treatment for heart failure with favorable effects, but its molecular mechanism has not been fully elucidated. Network pharmacological analysis and molecular docking revealed that Heat shock transcription factor 1 (HSF1) is a potential target of AS-IV. We designed cellular and animal experiments to investigate the role and intrinsic molecular mechanisms of AS-IV in ameliorating pressure overload-induced heart failure. In cellular experiments, Myocardial microvascular endothelial cells (MMVECs) were cultured in isolation and stimulated by adding high and low concentrations of AS-IV, and a cell model with down-regulation of HSF1 expression was constructed by using siRNA technology. Changes in the expression of key molecules of HSF1/VEGF signaling pathway and differences in tube-forming ability were detected in different groups of cells using PCR, WB and tube-forming assay. In animal experiments, TAC technology was applied to establish a pressure overload-induced heart failure model in C57 mice, postoperative mice were ingested AS-IV by gavage, and adenoviral transfection technology was applied to construct a mouse model with down-regulation of HSF1 expression.Small animal ultrasound for cardiac function assessment, MASSON staining, CD31 immunohistochemistry, and Western blotting (WB) were performed on the mice. The results showed that AS-IV could promote the expression of key molecules of HSF1/VEGF signaling pathway, enhance the tube-forming ability of MMVECs, increase the density of myocardial capillaries, reduce myocardial fibrosis, and improve the cardiac function of mice with TAC.AS-IV could modulate the HSF1/VEGF signaling pathway to promote the angiogenesis and improve the pressure overload-induced heart failure.

An Increase in HSF1 Expression Directs Human Mammary Epithelial Cells toward a Mesenchymal Phenotype.

HSF1 is a well-known heat shock protein expression regulator in response to stress. It also regulates processes important for growth, development or tumorigenesis. We studied the HSF1 influence on the phenotype of non-tumorigenic human mammary epithelial (MCF10A and MCF12A) and several triple-negative breast cancer cell lines. MCF10A and MCF12A differ in terms of HSF1 levels, morphology, growth in Matrigel, expression of epithelial (CDH1) and mesenchymal (VIM) markers (MCF10A are epithelial cells; MCF12A resemble mesenchymal cells). HSF1 down-regulation led to a reduced proliferation rate and spheroid formation in Matrigel by MCF10A cells. However, it did not affect MCF12A proliferation but led to CDH1 up-regulation and the formation of better organized spheroids. HSF1 overexpression in MCF10A resulted in reduced CDH1 and increased VIM expression and the acquisition of elongated fibroblast-like morphology. The above-mentioned results suggest that elevated levels of HSF1 may direct mammary epithelial cells toward a mesenchymal phenotype, while a lowering of HSF1 could reverse the mesenchymal phenotype to an epithelial one. Therefore, HSF1 may be involved in the remodeling of mammary gland architecture over the female lifetime. Moreover, HSF1 levels positively correlated with the invasive phenotype of triple-negative breast cancer cells, and their growth was inhibited by the HSF1 inhibitor DTHIB.

Mitochondrial HSF1 triggers mitochondrial dysfunction and neurodegeneration in Huntington's disease.

Aberrant localization of proteins to mitochondria disturbs mitochondrial function and contributes to the pathogenesis of Huntington's disease (HD). However, the crucial factors and the molecular mechanisms remain elusive. Here, we found that heat shock transcription factor 1 (HSF1) accumulates in the mitochondria of HD cell models, a YAC128 mouse model, and human striatal organoids derived from HD induced pluripotent stem cells (iPSCs). Overexpression of mitochondria-targeting HSF1 (mtHSF1) in the striatum causes neurodegeneration and HD-like behavior in mice. Mechanistically, mtHSF1 facilitates mitochondrial fission by activating dynamin-related protein 1 (Drp1) phosphorylation at S616. Moreover, mtHSF1 suppresses single-stranded DNA-binding protein 1 (SSBP1) oligomer formation, which results in mitochondrial DNA (mtDNA) deletion. The suppression of HSF1 mitochondrial localization by DH1, a unique peptide inhibitor, abolishes HSF1-induced mitochondrial abnormalities and ameliorates deficits in an HD animal model and human striatal organoids. Altogether, our findings describe an unsuspected role of HSF1 in contributing to mitochondrial dysfunction, which may provide a promising therapeutic target for HD.

The generation of PD-L1 and PD-L2 in cancer cells: From nuclear chromatin reorganization to extracellular presentation.

The immune checkpoint blockade (ICB) targeting on PD-1/PD-L1 has shown remarkable promise in treating cancers. However, the low response rate and frequently observed severe side effects limit its broad benefits. It is partially due to less understanding of the biological regulation of PD-L1. Here, we systematically and comprehensively summarized the regulation of PD-L1 from nuclear chromatin reorganization to extracellular presentation. In PD-L1 and PD-L2 highly expressed cancer cells, a new TAD (topologically associating domain) (chr9: 5,400,000-5,600,000) around CD274 and CD273 was discovered, which includes a reported super-enhancer to drive synchronous transcription of PD-L1 and PD-L2. The re-shaped TAD allows transcription factors such as STAT3 and IRF1 recruit to PD-L1 locus in order to guide the expression of PD-L1. After transcription, the PD-L1 is tightly regulated by miRNAs and RNA-binding proteins via the long 3'UTR. At translational level, PD-L1 protein and its membrane presentation are tightly regulated by post-translational modification such as glycosylation and ubiquitination. In addition, PD-L1 can be secreted via exosome to systematically inhibit immune response. Therefore, fully dissecting the regulation of PD-L1/PD-L2 and thoroughly detecting PD-L1/PD-L2 as well as their regulatory networks will bring more insights in ICB and ICB-based combinational therapy.

Molecular characterization of Hsf1 as a master regulator of heat shock response in the thermotolerant methylotrophic yeast Ogataea parapolymorpha.

Ogataea parapolymorpha (Hansenula polymorpha DL-1) is a thermotolerant methylotrophic yeast with biotechnological applications. Here, O. parapolymorpha genes whose expression is induced in response to heat shock were identified by transcriptome analysis and shown to possess heat shock elements (HSEs) in their promoters. The function of O. parapolymorpha HSF1 encoding a putative heat shock transcription factor 1 (OpHsf1) was characterized in the context of heat stress response. Despite exhibiting low sequence identity (26%) to its Saccharomyces cerevisiae homolog, OpHsf1 harbors conserved domains including a DNA binding domain (DBD), domains involved in trimerization (TRI), transcriptional activation (AR1, AR2), transcriptional repression (CE2), and a C-terminal modulator (CTM) domain. OpHSF1 could complement the temperature sensitive (Ts) phenotype of a S. cerevisiae hsf1 mutant. An O. parapolymorpha strain with an H221R mutation in the DBD domain of OpHsf1 exhibited significantly retarded growth and a Ts phenotype. Intriguingly, the expression of heat-shock-protein-coding genes harboring HSEs was significantly decreased in the H221R mutant strain, even under non-stress conditions, indicating the importance of the DBD for the basal growth of O. parapolymorpha. Notably, even though the deletion of C-terminal domains (ΔCE2, ΔAR2, ΔCTM) of OpHsf1 destroyed complementation of the growth defect of the S. cerevisiae hsf1 strain, the C-terminal domains were shown to be dispensable in O. parapolymorpha. Overexpression of OpHsf1 in S. cerevisiae increased resistance to transient heat shock, supporting the idea that OpHsf1 could be useful in the development of heat-shock-resistant yeast host strains.

Combined inhibition of AURKA and HSF1 suppresses proliferation and promotes apoptosis in hepatocellular carcinoma by activating endoplasmic reticulum stress.

PURPOSE: In this study we aimed to assess the anti-tumor effect of co-inhibition of Aurora kinase A (AURKA) and heat shock transcription factor 1 (HSF1) on hepatocellular carcinoma (HCC), as well as to explore the mechanism involved. METHODS: Expression of AURKA and HSF1 in primary HCC tissues and cell lines was detected by immunohistochemistry (IHC), qRT-PCR and Western blotting. AURKA was knocked down in HepG2 and BEL-7402 HCC cells using lentivirus-mediated RNA interference. Next, CCK-8, clone formation, transwell and flow cytometry assays were used to assess their viability, migration, invasion and apoptosis, respectively. The expression of proteins related to cell cycle progression, apoptosis and endoplasmic reticulum stress (ERS) was analyzed using Western blotting. In addition, in vivo tumor growth of HCC cells was assessed using a nude mouse xenograft model, and the resulting tumors were evaluated using HE staining and IHC. RESULTS: Both AURKA and HSF1 were highly expressed in HCC tissues and cells, while being negatively related to HCC prognosis. Knockdown of AURKA significantly inhibited the colony forming and migrating capacities of HCC cells. In addition, we found that treatment with an AURKA inhibitor (Danusertib) led to marked reductions in the proliferation and migration capacities of the HCC cells, and promoted their apoptosis. Notably, combined inhibition of AURKA and HSF1 induced HCC cell apoptosis, while increasing the expression of ERS-associated proteins, including p-eIF2α, ATF4 and CHOP. Finally, we found that co-inhibition of AURKA and HSF1 elicited an excellent in vivo antitumor effect in a HCC mouse model with a relatively low cytotoxicity. CONCLUSIONS: Combined inhibition of AURKA and HSF1 shows an excellent anti-tumor effect on HCC cells in vitro and in vivo, which may be mediated by ERS. These findings suggest that both AURKA and HSF1 may serve as targets for HCC treatment.

Hypoxia-Induced Cancer Cell Responses Driving Radioresistance of Hypoxic Tumors: Approaches to Targeting and Radiosensitizing.

Within aggressive malignancies, there usually are the "hypoxic zones"-poorly vascularized regions where tumor cells undergo oxygen deficiency through inadequate blood supply. Besides, hypoxia may arise in tumors as a result of antiangiogenic therapy or transarterial embolization. Adapting to hypoxia, tumor cells acquire a hypoxia-resistant phenotype with the characteristic alterations in signaling, gene expression and metabolism. Both the lack of oxygen by itself and the hypoxia-responsive phenotypic modulations render tumor cells more radioresistant, so that hypoxic tumors are a serious challenge for radiotherapy. An understanding of causes of the radioresistance of hypoxic tumors would help to develop novel ways for overcoming this challenge. Molecular targets for and various approaches to radiosensitizing hypoxic tumors are considered in the present review. It is here analyzed how the hypoxia-induced cellular responses involving hypoxia-inducible factor-1, heat shock transcription factor 1, heat shock proteins, glucose-regulated proteins, epigenetic regulators, autophagy, energy metabolism reprogramming, epithelial-mesenchymal transition and exosome generation contribute to the radioresistance of hypoxic tumors or may be inhibited for attenuating this radioresistance. The pretreatments with a multitarget inhibition of the cancer cell adaptation to hypoxia seem to be a promising approach to sensitizing hypoxic carcinomas, gliomas, lymphomas, sarcomas to radiotherapy and, also, liver tumors to radioembolization.

Regulatory effect of heat shock transcription factor-1 gene on heat shock proteins and its transcriptional regulation analysis in small abalone Haliotis diversicolor.

BACKGROUND: The effects of diverse stresses ultimately alter the structures and functions of proteins. As molecular chaperones, heat shock proteins (HSPs) are a group of highly conserved proteins that help in the refolding of misfolded proteins and the elimination of irreversibly damaged proteins. They are mediated by a family of transcription factors called heat shock factors (HSFs). The small abalone Haliotis diversicolor is a species naturally distributed along the southern coast of China. In this study, the expression of HdHSF1 was inhibited by RNAi in hemocytes in order to further elucidate the regulatory roles of HdHSF1 on heat shock responsive genes in abalone. Meanwhile, to understand the transcriptional regulation of the HdHSF1 gene, the 5'-upstream regulatory region of HdHSF1 was characterized, and the relative promoter activity was examined by dual-luciferase reporter gene assay system in HEK293T cell lines. RESULTS: After the inhibition of the H. diversicolor HSF1 gene (HdHSF1) by dsRNA (double-stranded RNA), the expression of most heat shock related-genes was down-regulated (p < 0.05). It indicated the importance of HdHSF1 in the heat shock response of H. diversicolor. Meanwhile, 5'-flanking region sequence (2633 bp) of the HdHSF1 gene was cloned; it contained a putative core promoter region, TATA box, CAAT box, CpG island, and many transcription elements. In HEK293T cells, the 5'-flanking region sequence can drive expression of the enhanced green fluorescent protein (EGFP), proving its promoter function. Exposure of cells to the high-temperature (39 °C and 42 °C) resulted in the activation of HdHSF1 promoter activity, which may explain why the expression of the HdHSF1 gene participates in heat shock response. Luciferase activity of different recombinant plasmids, which contained different truncated promoter fragments of the HdHSF1 gene in HEK293T cells, revealed the possible active regions of the promoter. To further identify the binding site of the critical transcription factor in the region, an expression vector with the site-directed mutation was constructed. After being mutated on the GATA-1 binding site, we found that the luciferase activity was significantly increased, which suggested that the GATA-1 binding site has a certain weakening effect on the activity of the HdHSF1 promoter. CONCLUSIONS: These findings suggest that GATA-1 may be one of the transcription factors of HdHSF1, and a possible signaling pathway mediated by HdHSF1 may exist in H. diversicolor to counteract the adverse effects of heat shock stress.