Inhibition Of HSF1 Research Articles

Insights from yeast into whether the inhibition of heat shock transcription factor (Hsf1) by rapamycin can prevent the Hsf1 activation that results from treatment with an Hsp90 inhibitor.

In human cells TORC1 mTOR (target of rapamycin) protein kinase complex renders heat shock transcription factor 1 (Hsf1) competent for stress activation. In such cells, as well as in yeast, the selective TORC1 inhibitor rapamycin blocks this activation in contrast to Hsp90 inhibitors which potently activate Hsf1. Potentially therefore rapamycin could prevent the Hsf1 activation that frequently compromises the efficiency of Hsp90 inhibitor cancer drugs. Little synergy was found between the effects of rapamycin and the Hsp90 inhibitor radicicol on yeast growth. However certain rapamycin resistance mutations sensitised yeast to Hsp90 inhibitor treatment and an Hsp90 mutation that overactivates Hsf1 sensitised cells to rapamycin. Rapamycin inhibition of the yeast Hsf1 was abolished by this Hsp90 mutation, as well as with the loss of Ppt1, the Hsp90-interacting protein phosphatase that is the ortholog of mammalian PP5. Unexpectedly Hsf1 activation was found to have a requirement for the rapamycin binding immunophilin FKBP12 even in the absence of rapamycin, while TORC1 "bypass" strains revealed that the rapamycin inhibition of yeast Hsf1 is not exerted through two of the major downstream targets of TORC1, the protein phosphatase regulator Tap42 and the protein kinase Sch9--the latter the ortholog of human S6 protein kinase 1. A problem with most of the Hsp90 inhibitor drugs now in cancer clinic trials is that they potently activate Hsf1. This leads to an induction of heat shock proteins, many of which have a "pro-survival" role in that they help to protect cells from apopotosis. As the activation of Hsf1 requires TORC1, inhibitors of mTOR kinase could potentially block this activation of Hsf1 and be of value when used in combination drug therapies with Hsp90 inhibitors. However many of the mechanistic details of the TORC1 regulation of Hsf1, as well as the interplay between cellular resistances to rapamycin and to Hsp90 inhibitors, still remain to be resolved.

Molecular Stress-inducing Compounds Increase Osteoclast Formation in a Heat Shock Factor 1 Protein-dependent Manner

Many anticancer therapeutic agents cause bone loss, which increases the risk of fractures that severely reduce quality of life. Thus, in drug development, it is critical to identify and understand such effects. Anticancer therapeutic and HSP90 inhibitor 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) causes bone loss by increasing osteoclast formation, but the mechanism underlying this is not understood. 17-AAG activates heat shock factor 1 (Hsf1), the master transcriptional regulator of heat shock/cell stress responses, which may be involved in this negative action of 17-AAG upon bone. Using mouse bone marrow and RAW264.7 osteoclast differentiation models we found that HSP90 inhibitors that induced a heat shock response also enhanced osteoclast formation, whereas HSP90 inhibitors that did not (including coumermycin A1 and novobiocin) did not affect osteoclast formation. Pharmacological inhibition or shRNAmir knockdown of Hsf1 in RAW264.7 cells as well as the use of Hsf1 null mouse bone marrow cells demonstrated that 17-AAG-enhanced osteoclast formation was Hsf1-dependent. Moreover, ectopic overexpression of Hsf1 enhanced 17-AAG effects upon osteoclast formation. Consistent with these findings, protein levels of the essential osteoclast transcription factor microphthalmia-associated transcription factor were increased by 17-AAG in an Hsf1-dependent manner. In addition to HSP90 inhibitors, we also identified that other agents that induced cellular stress, such as ethanol, doxorubicin, and methotrexate, also directly increased osteoclast formation, potentially in an Hsf1-dependent manner. These results, therefore, indicate that cellular stress can enhance osteoclast differentiation via Hsf1-dependent mechanisms and may significantly contribute to pathological and therapeutic related bone loss.

Triptolide-induced Cell Death in Pancreatic Cancer Is Mediated by O-GlcNAc Modification of Transcription Factor Sp1

Pancreatic cancer, the fourth most prevalent cancer-related cause of death in the United States, is a disease with a dismal survival rate of 5% 5 years after diagnosis. One of the survival proteins responsible for its extraordinary ability to evade cell death is HSP70. A naturally derived compound, triptolide, and its water-soluble prodrug, Minnelide, down-regulate the expression of this protein in pancreatic cancer cells, thereby causing cell death. However, the mechanism of action of triptolide has not been elucidated. Our study shows that triptolide-induced down-regulation of HSP70 expression is associated with a decrease in glycosylation of the transcription factor Sp1. We further show that triptolide inhibits glycosylation of Sp1, inhibiting the hexosamine biosynthesis pathway, particularly the enzyme O-GlcNAc transferase. Inhibition of O-GlcNAc transferase prevents nuclear localization of Sp1 and affects its DNA binding activity. This in turn down-regulates prosurvival pathways like NF-κB, leading to inhibition of HSF1 and HSP70 and eventually to cell death. In this study, we evaluated the mechanism by which triptolide affects glycosylation of Sp1, which in turn affects downstream pathways controlling survival of pancreatic cancer cells.

A NOVEL APPROACH OF VIROTHERAPY BASED HSF-1 shRNA IN CANCER ERADICATION

Cancer is the second leading cause of death worldwide with continues rise mortality rate. Current cancer treatment modalities are still ineffective and associated with many side effects leading to robust research to find new specific target therapy. Heat shock factor (HSF)-1 is heat shock response mediator protein and act as transcription factor for HSP encoding gene. Many cancers have up-regulated HSP as a result of increase HSF-1 expression. Interestingly, inhibition of HSF-1 has no effect to normal cell, indicating HSF-1 as promises target therapy. RNAi is potential mechanism to block and down regulate HSF-1 which will affect many cellular processes in cancer cell. Combining RNAi base treatment with oncolityc viruses will boost the therapeutic effect of this novel treatment. Despite its potency, this modality still need further research in order to evaluate its efficacy and optimal doses to gain optimal result.

Proteotoxic stress targeted therapy (PSTT)

About a decade ago a multiple myeloma patient demonstrated a complete response to a proteasome inhibitor Bortezomib. This case generated a lot of hopes, and after successful clinical trials Bortezomib was approved by FDA and became the drug of choice for this disease. A high sensitivity of multiple myeloma to proteasome inhibition is probably related to extensive production immunoglobulins by the cancer cells. Bortezomib prevents degradation of these proteins, which accumulate and cause a strong ER stress, eventually triggering apoptosis [1, 2]. Unfortunately, many patients show only partial response to Bortezomib, in part due to the development of resistance [3]. Among multiple mechanism of resistance to Bortezomib, cells upregulate expression of molecular chaperones, e.g. Hsp27, which protect from the buildup of abnormal protein species [4-7]. The balance between toxicity of abnormal polypeptides and protective function of chaperones defines the outcome of the treatment with proteasome inhibitors. Bortezomib has also been tested in trials for treatment of solid tumors, either alone or in combination with other drugs, and results have been promising. The selective sensitivity of various cancer cells to proteasome inhibitors as compared to normal cells is probably because cancer cells have to handle higher levels of abnormal polypeptides, since they are exposed to adverse conditions of tumor microenvironments, oxidative stress and because of genetic instability and associated accumulation of mutant proteins [8]. Accordingly, cancer cells often have increased levels of molecular chaperones, like Hsp72 or Hsp27, as well as the heat shock transcription factor Hsf1 [9]. Furthermore, it was found that cancers have special requirements for these factors, since Hsf1 and hsp72 knockout mice demonstrate dramatic resistance to certain types of cancer, e.g. skin or breast cancer [10-12]. These data led to a concept of the “non-oncogene addiction” of cancers to Hsf1 and molecular chaperones, and predicted that inhibition of chaperones could be a potent novel approach towards development of therapeutics [8]. In fact, there have been number of publications that pharmacological inhibition or siRNA-mediated depletion of Hsf1, Hsp72 or Hsp27 can selectively cause apoptosis or growth inhibition in various types of cancer [9, 13]. Furthermore, anti-sense RNA against Hsp27 has been tested and demonstrated promising results with bladder cancer [14, 15]. Since downregulation of Hsf1 or chaperones increases the generation of abnormal proteins, while inhibition of proteasome blocks their degradation, the combination of these treatments could synergistically precipitate a selective demise of cancer cells. Indeed, it was demonstrated that downregulation or inhibition of Hsf1 can potently enhance sensitivity of cancer cells to Bortezomib [16, 17]. Accordingly, a lot of efforts and resources have been invested in academic labs and industry in development of inhibitors of Hsf1 and various chaperones, but so far there have been no breakthroughs. The paper by Neznanov et al. [18] offers an alternative approach towards sensitization of cancer cells to proteasome inhibitors. Instead of inhibition of chaperones, this group proposes to cause a buildup of abnormal polypeptides by heat shock. They demonstrated that a mild non-toxic heat shock strongly enhances apoptosis caused by sub-toxic concentrations of Bortezomib. Although there are no data on the effects of heat shock beyond cell culture models, an interesting possibility is that a combination of local hyperthermia and Bortezomib could be beneficial for treatment of localized tumors. Furthermore, since only mild heat treatment was necessary for the synergistic effect with Bortezomib, there is a possibility that the fever-range temperature, which could be triggered by pyrogenes can be sufficient for enhancement of Bortezomib-induced anti-cancer effects. This possibility opens up a range of approaches towards treatment. As an alternative way of generating abnormal proteins, the authors used puromycin. This inhibitor accepts growing polypeptide chains, and aborts further growth, leading to the release of incomplete chains, which of course cannot fold properly and are toxic. At low sub-toxic concentrations, puromycin could strongly potentiate killing of cancer cells by Bortezomib. In this set of experiments, the authors tested not only cell culture models, but also a mouse model of multiple myeloma. They demonstrated that Bortezomib alone only partially reduced growth of multiple myeloma tumors transplanted into syngenic animals. On the other hand, a combination of Bortezomib and low doses of puromycin led to almost complete inhibition. Overall, the combination of proteasome inhibitors with puromycin, heat shock or other treatments that generate a buildup of abnormal polypeptides represents a novel, and potentially powerful approach towards cancer treatment.

A novel HSP90 modulator with selective activity against thyroid cancers in vitro

Heat shock protein 90 (HSP90) is a chaperone protein regulating several client proteins involved in thyroid cancer development. The purpose of this study was to mechanistically evaluate a novel, natural product drug with anticancer activity in thyroid cancer cell lines in vitro for future translational applications. A total of 285 natural plant extracts and compounds were evaluated for anticancer activity by MTS assay. Apoptosis and cell cycle arrest were characterized by annexin V-propidium iodide (PI) flow cytometry. HSP90 and client protein modulation, as well as apoptosis confirmation, as demonstrated by Western blot analysis. Of the 285 compounds and products tested, 45 demonstrated antiproliferative activity in thyroid cancers by MTS assay. BTIMNP_D004 demonstrated the greatest inhibition (IC(50) = 155-2,890 nM in thyroid cancers). Activity was cancer-cell selective compared to fibroblasts, with increased potency over 17-AAG in BCPAP, FTC133, and DRO81-1 cells. D004 modulated cell cycle arrest after 18 hours (G(1)/G(0) --> S and G(2)/M) with 30% FTC133 cells shifted, 22% BCPAP cells shifted, and 15% SW1736 cells shifted versus controls (P < .01, P < .01, and P < .05, respectively). A total of 1 muM D004 induced significant apoptosis, with 76% BCPAP cells gated after 18 hours (annexin V-PI staining vs <3% in controls, P < .01; and 80% FTC133 cells vs 4% controls; P < .01). Western blot analysis demonstrated modulation of HSP90 expression levels, with inhibition of HSF-1, AKT, and caspase-3 expression, and cleavage of PARP in both BCPAP and FTC133 cells. BTIMNP_D004 is a novel natural product drug with anticancer activity against thyroid cancers in vitro, and may act through induction of apoptosis, modulation of cell cycle arrest, and modulation of heat shock chaperone proteins including HSP90. These preliminary in vitro data support future preclinical studies for translational applications.

Yeast Yak1 kinase, a bridge between PKA and stress‐responsive transcription factors, Hsf1 and Msn2/Msn4

Hsf1 and Msn2/Msn4 transcription factors in Saccharomyces cerevisiae play important roles in cellular homeostasis by activating gene expression in response to multiple stresses including heat shock, oxidative stress and nutrient starvation. Although it has been known that nuclear import of Msn2 is inhibited by PKA-dependent phosphorylation, the mechanism for PKA-dependent regulation of Hsf1 is not well understood. Here we demonstrate that Yak1 kinase, which is under the negative control of PKA, activates both Hsf1 and Msn2 by phosphorylation when PKA activity is lowered by glucose depletion or by overexpressing Pde2 that hydrolyses cAMP. We show that Yak1 directly phosphorylates Hsf1 in vitro, leading to the increase in DNA binding activity of Hsf1. We also demonstrate that Yak1 phosphorylates Msn2 in vitro, but does not affect DNA binding activity of Msn2 or nuclear localization of Msn2 upon glucose depletion. These results suggest a central role for Yak1 in mediating PKA-dependent inhibition of Hsf1 and Msn2/Msn4.

Involvement of heat shock factor 1 in statin-induced transcriptional upregulation of endothelial thrombomodulin.

Statins upregulate endothelial thrombomodulin (TM) by mechanisms that involve members of the Kruppel-like factor family. Although Kruppel-like factors are unequivocally implicated in this process, experimental evidence points to additional mechanisms. Deletion/mutation analysis of reporter constructs was used to demonstrate that mutation of the SP1/Kruppel-like factor element in the TM promoter only partially abolishes statin-induced TM upregulation, whereas simultaneous mutation of relevant heat shock elements and SP1/Kruppel-like factor element completely prevents statin-induced TM upregulation, thus demonstrating a role for heat shock factors (HSFs). We further identified the pathway by which statins increase binding of HSF1 to heat shock elements in the TM promoter. Specifically, statins caused NO-dependent dissociation of HSF1 from heat shock protein 90, nuclear translocation of HSF1, and binding to heat shock elements in the TM promoter. Statins also decreased nuclear content of the HSF1 chaperone 14-3-3beta. In addition to reducing TM upregulation, inhibition of HSF1 reduced statin-induced upregulation of tissue plasminogen activator, whereas downregulation of thrombomospondin, plasminogen activator inhibitor 1, or connective tissue growth factor was unaffected. Knockdown of 14-3-3beta or inhibition of HSF1 phosphorylation enhanced the effect of statins on TM and tissue plasminogen activator, but did not influence thrombomospondin, plasminogen activator inhibitor 1, or connective tissue growth factor. These data demonstrate that HSF1 is involved in statin-induced regulation of TM. They also suggest that analogous mechanisms may apply to genes that are upregulated by statins, but not to downregulated genes. These results may have broad implications and suggest the use of heat shock protein modulators to selectively regulate pleiotropic statin effects.

Genome-wide analysis of human HSF1 signaling reveals a transcriptional program linked to cellular adaptation and survival

Although HSF1 plays an important role in the cellular response to proteotoxic stressors, little is known about the structure and function of the human HSF1 signaling network under both stressed and unstressed conditions. In this study, we used a combination of chromatin immunoprecipitation microarray analysis and time course gene expression microarray analysis with and without siRNA-mediated inhibition of HSF1 to comprehensively identify genes regulated directly and indirectly by HSF1. The correlation between promoter binding and gene expression was not significant for all genes bound by HSF1, suggesting that HSF1 binding per se is not sufficient for expression. However, the correlation with promoter binding was significant for genes identified as HSF1-regulated following siRNA knockdown. Among promoters bound by HSF1 following heat shock, a gene ontology analysis showed significant enrichment only in categories related to protein folding. In contrast, analysis of the extended HSF1 signaling network following siRNA knockdown showed enrichment in a variety of categories related to protein folding, anti-apoptosis, RNA splicing, ubiquitinylation and others, highlighting a complex transcriptional program regulated directly and indirectly by HSF1.

Reduced tolerance of immature renal tubules to anoxia by HSF-1 decoy.

Immature animals demonstrate an amplified heat shock response following a variety of insults compared with that seen in mature animals (M). The potential role of the heat shock response in modulating immature tolerance to injury was compared between rat pups, 10 postnatal days of age (P10), and M. Baseline levels of the heat shock transcription factor (HSF-1) were substantially elevated in P10 compared with M animals. In uninjured P10 pups, HSF-1 level was comparable to that of M animals subjected to 45 min of ischemia. As anticipated, the integrity of suspensions of tubules exposed to anoxia was preserved in P10 animals (23% LDH release) compared with M (40%), P < 0.01. The effect of targeted inhibition of HSF-1 on tubular integrity was studied using a cyclic oligonucleotide decoy. The HSF-1 decoy increased the severity of anoxic injury in P10 pups to a level comparable with M animals. LDH release was 33% in decoy-treated P10 tubules compared with 40% in M. When P10 tubules were treated with scrambled decoy, resistance to anoxia remained intact (24%). The increased vulnerability of the tubular suspension to injury was specific to the HSF-1 decoy and proportional to the dose of decoy applied. This study demonstrates maturation in the abundance of HSF-1 in the immature rat kidney. The loss of resistance of immature tubules to anoxia with specific inhibition of HSF-1 may be due to its effect on the heat shock response or other signaling pathways of critical pathobiological importance in renal cell injury.