Phosphorylation Of Heat Shock Factor Research Articles

Distinct induction pathways of heat shock protein 27 in human keratinocytes: Heat stimulation or capsaicin through phosphorylation of heat shock factor 1 at serine 326 and/or suppression of ΔNp63

Epidermal keratinocytes, forming the outermost layer of the human body, serve as a crucial barrier against diverse external stressors such as ultraviolet radiation. Proper keratinocyte differentiation and effective responses to external stimuli are pivotal for maintaining barrier integrity. Heat is one such stimulus that triggers the synthesis of heat shock proteins (HSPs) when cells are exposed to temperatures above 42 °C. Additionally, activation of the transient receptor potential cation channel subfamily V member 1 (TRPV1) occurs at 42 °C. Here, we explore the interplay between TRPV1 signaling and HSP induction in human keratinocytes. Both heat and capsaicin, a TRPV1 agonist, induce expression of HSP27, HSP70, and HSP90 in keratinocytes. Interestingly, pharmacological inhibition of TRPV1 attenuates heat-induced HSP27 expression, but not that of HSP70 or HSP90. Furthermore, both heat and capsaicin stimulation result in distinct phosphorylation patterns of heat shock factor 1 (HSF1), with phosphorylation at serine 326 being a common feature. Notably, genetic manipulation to mimic dephosphorylation of HSF1 at serine 326 reduces HSP27 levels. Additionally, ΔNp63, a key regulator of epidermal differentiation, negatively modulates HSP27 expression independently of HSF1 phosphorylation status. While heat stimulation has no effect on ΔNp63 expression, capsaicin reduces its levels. The precise role of TRPV1 signaling in keratinocytes warrants further investigation for a comprehensive understanding of its impact on barrier function.

Abstract 4319: HSF1 inhibition induces pancreatic ductal adenocarcinoma autophagy through JNK

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related mortality, with a 5-year survival rate of 12%. Chemoresistance due to autophagy, a natural process of proteotoxic stress response, is the most common chemotherapy challenge for pancreatic cancer. Autophagy, which maintains cellular homeostasis by removing damaged or unnecessary proteins and organelles, is a self-degradation process regulated by the mammalian target of rapamycin complex (mTORC1). Excessive levels of autophagy can be detrimental and lead to cell death. Nevertheless, this dual function of autophagy in regulating pancreatic cancer remains poorly understood. It has been reported that heat shock factor 1 (HSF1), a critical pro-oncogenic transcription factor in the proteotoxic stress response, inhibits c-Jun N-terminal kinase 1/2 (JNK1/2) signaling pathway to maintain mTORC1 activity and control cell size. HSF1 is highly expressed in pancreatic tumors, and inhibition of HSF1 via small molecules enhances the chemotherapy drug effect on PDAC programmed cell death. However, the PDAC autophagy response in HSF1 inhibition is currently not fully understood. Objective: The objective of this study is to investigate the molecular mechanism of HSF1 inhibition in PDAC autophagy. Methods and Results: In human PDAC cells, Western blotting analysis revealed that HSF1 inhibition via small molecular including CCT361814/NXP800, an HSF1 inhibitor in phase I clinical trials, increased autophagy marker microtubule-associated protein 1A/1B-light chain 3 (LC3) lipidation and decreased expression of phosphorylation of HSF1 at Ser326 and total HSF1 protein, conversely using HSF1 overexpression model reversed this effect. Inhibition of autophagy via 3-methyladenine reversed HSF1 inhibition-induced autophagy. Results from transmission electron microscopy and GFP-LC3 reporter revealed that HSF1 inhibition induced autophagosome formation and accumulation. Furthermore, HSF1 inhibition induced phosphorylation of JNK1/2 at Thr183/Tyr185 and decreased mTORC1 activity, while knockdown JNK1/2 diminished HSF1 inhibition-induced LC3 lipidation. The combination of chemotherapy drugs with HSF1 inhibitors decreased PDAC cell viability. Additionally, blocking autophagy increased HSF1 inhibition-induced apoptosis. Conclusions: In conclusion, JNK1/2 is involved in HSF1 inhibition-induced PDAC autophagy, targeting autophagy could unveil a novel treatment strategy for pancreatic cancer through targeted HSF1 inhibition. Citation Format: Shruti Ghai, Alex Young, Kuo-Hui Su. HSF1 inhibition induces pancreatic ductal adenocarcinoma autophagy through JNK [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4319.

MnO2 nanoparticles trigger hepatic lipotoxicity and mitophagy via mtROS-dependent Hsf1Ser326 phosphorylation

Manganese (Mn) is an essential element for maintaining normal metabolism in vertebrates. Mn dioxide nanoparticles (MnO2 NPs), a novel Mn source, have shown great potentials in biological and biomedical applications due to their distinct physical and chemical properties. However, little is known about potential adverse effects on animal or cellular metabolism. Here, we investigated whether and how dietary MnO2 NPs affect hepatic lipid metabolism in vertebrates. We found that, excessive MnO2 NPs intake increased hepatic and mitochondrial Mn content, promoted hepatic lipotoxic disease and lipogenesis, and inhibited hepatic lipolysis and fatty acid β-oxidation. Moreover, excessive MnO2 NPs intake induced hepatic mitochondrial oxidative stress, damaged mitochondrial function, disrupted mitochondrial dynamics and activated mitophagy. Importantly, we uncovered that mtROS-activated phosphorylation of heat shock factor 1 (Hsf1) at Ser326 residue mediated MnO2 NPs-induced hepatic lipotoxic disease and mitophagy. Mechanistically, MnO2 NPs-induced lipotoxicity and mitophagy were via mtROS-induced phosphorylation and nucleus translocation of Hsf1 and its DNA binding capacity to plin2/dgat1 and bnip3 promoters, respectively. Overall, our findings uncover novel mechanisms by which mtROS-mediated mitochondrial dysfunction and phosphorylation of Hsf1S326 contribute to MnO2 NPs-induced hepatic lipotoxicity and mitophagy, which provide new insights into the effects of metal oxides nanoparticles on hepatotoxicity in vertebrates.

Heat shock factor 1 drives regulatory T-cell induction to limit murine intestinal inflammation

The heat shock response is a critical component of the inflammatory cascade that prevents misfolding of new proteins and regulates immune responses. Activation of CD4+ T cells causes an upregulation of heat shock transcription factor, heat shock factor 1 (HSF1). We hypothesized that HSF1 promotes a pro-regulatory phenotype during inflammation. To validate this hypothesis, we interrogated cell-specific HSF1 knockout mice and HSF1 transgenic mice using in vitro and in vivo techniques. We determined that while HSF1 expression was induced by anti-CD3 stimulation alone, the combination of anti-CD3 and TGFβ, a vital cytokine for regulatory T cell (Treg) development, resulted in increased activating phosphorylation of HSF1, leading to increased nuclear translocation and binding to heat shock response elements. Using ChIP, we demonstrate direct binding of HSF1 to foxp3 in isolated murine CD4+ T cells, which in turn coincided with induction of FoxP3 expression. We defined that conditional knockout of HSF1 decreased development and function of Tregs and overexpression of HSF1 led to increased expression of FoxP3 along with enhanced Treg suppressive function. Adoptive transfer of CD45RBHigh CD4 colitogenic T cells along with HSF1 transgenic CD25+ Tregs prevented intestinal inflammation when WT Tregs did not. Finally, overexpression of HSF1 provided enhanced barrier function and protection from murine ileitis. This study demonstrates that HSF1 promotes Treg development and function and may represent both a crucial step in the development of induced regulatory T cells and an exciting target for the treatment of inflammatory diseases with a regulatory T cell component.

Kainic Acid-Induced Excitotoxicity Leads to the Activation of Heat Shock Response.

Heat shock response (HSR) which is regulated by heat shock factor 1 (HSF1) is the most important mechanism and the major regulator that prevents protein aggregation in neurodegenerative diseases. Excitotoxicity, which is the accumulation of excess glutamate in synaptic cleft, is observed in age-dependent neurodegenerative diseases and also in stroke, epilepsy, and brain trauma. Only a few studies in the literature show the link between excitotoxicity and HSR. In this study, we aimed to show the molecular mechanism underlying this link. We applied heat shock (HS) treatment and induced excitotoxicity with kainic acid (KA) in neuroblastoma (SHSY-5Y) and glia (immortalized human astrocytes (IHA)) cells. We observed that, only in SHSY-5Y cells, heat shock preconditioning increases cell survival after KA treatment. GLT-1 mRNA expression is increased as a result of KA treatment and HS due to the elevation of HSF1 binding to GLT-1 promoter which was induced by HSF1 phosphorylation and sumolation in SHSY-5Y cells. Additionally, glutamine synthetase and glutaminase expressions are increased after HS preconditioning in SHSY-5Y cells indicating that HS activates glutamate metabolism modulators and accelerates the glutamate cycle. In glia cells, we did not observe the effect of HS preconditioning. In summary, heat shock preconditioning might be protective against excitotoxicity-related cell death and degeneration.

Heat shock factor 1 is a promising therapeutic target against adult T-cell leukemia

Patients with adult T-cell leukemia (ATL), which is caused by human T-cell leukemia virus type 1 (HTLV-1), show poor prognosis because of drug resistance. Heat shock protein (HSP) 90 is reportedly essential for ATL cell survival as it regulates important signaling pathways, thereby making HSP90 inhibitors new therapeutic candidates for ATL. However, HSP90 inhibition increases the expression of other HSPs, suggesting that HSPs may contribute to drug resistance. The heat shock factor 1 (HSF1) transcription factor is the primary regulator of the expression of HSPs. Furthermore, targeting HSF1 disrupts the HSP90 chaperone function. Herein, we demonstrated that HSF1 is overexpressed in HTLV-1-infected T cells. HSF1 knockdown inhibited the proliferation of HTLV-1-infected T cells. HSF1 inhibitor KRIBB11 reduced the expression and phosphorylation of HSF1, downregulated HSP70 and HSP27 expression, and suppressed Akt, nuclear factor-κB, and AP-1 signals. KRIBB11 treatment induced DNA damage, upregulated p53 and p21, and reduced the expression of cyclin D2/E, CDK2/4, c-Myc, MDM2, and β-catenin, thereby preventing retinoblastoma protein phosphorylation and inhibiting G1-S cell cycle progression. KRIBB11 also induced caspase-mediated apoptosis concomitant with the suppression of Bcl-xL, Mcl-1, XIAP, c-IAP1/2, and survivin expression. KRIBB11 inhibited HSP70 and HSP90 upregulation through treatment with AUY922, an HSP90 inhibitor, and enhanced the cytotoxic effect of AUY922, suggesting a salvage role of HSF1-dependent HSP induction in response to drug treatment. Finally, treatment of mice with KRIBB11 reduced ATL tumor growth. Therefore, this study provides a strong rationale to target HSF1 and validates the anti-ATL activity of KRIBB11.

N-butyrate increases heat shock protein 70 through heat shock factor 1 and AMP-activated protein kinase pathways in human intestinal Caco-2 cells

Impaired integrity of the intestinal epithelium is a cause of intestinal and extraintestinal diseases. Heat shock protein 70 (HSP70), a cytoprotective protein, plays an important role in maintaining intestinal homeostasis. The intestinal expression of HSP70 is linked with the local microbiota. The present study investigated the molecular mechanisms underlying the upregulation of HSP70 by n-butyrate, a major metabolite of the intestinal microbiota in human intestinal Caco-2 cells. Treatment of Caco-2 cells with n-butyrate upregulated HSP70 protein and mRNA levels in a dose-dependent manner. Using luciferase reporter assay, it was found that n-butyrate enhanced the transcriptional activity of HSP70. These effects were sensitive to the inhibition of heat shock factor 1 (HSF1), a transcription factor, and AMP-activated protein kinase (AMPK). N-butyrate increased the phosphorylation (activity) of HSF1 and AMPK. Taken together, this study shows that n-butyrate is partly involved in the microbiota-dependent intestinal expression of HSP70, and the effect is exerted through the HSF1 and AMPK pathways.

Cisplatin-induced HSF1-HSP90 axis enhances the expression of functional PD-L1 in oral squamous cell carcinoma.

Immune checkpoint inhibitor-based cancer immunotherapy has provided an additional therapeutic option for oral squamous cell carcinoma (OSCC) with recurrence or distant metastases. However, further improvement of OSCC treatment is required to develop the optimal combination or order for chemoradiotherapy and immunotherapy. Along with the accumulation of clinical knowledge and evidence, it is also essential to clarify the biological impact of chemo-radiotherapeutic agents on the cancer immune microenvironment. In this study, we investigated the effects of cisplatin (CDDP), a key therapeutic agent for OSCC, on programmed death-ligand 1 (PD-L1) expression in OSCC lines. Although CDDP treatment increased the surface levels of PD-L1 on OSCC cell lines, the gene and total protein expression levels of PD-L1 were not altered. We also demonstrated that the phosphorylation of heat shock factor 1 and heat shock protein 90 was involved in this process. In addition, CDDP-induced PD-L1 attenuated the target-specific cytotoxic T lymphocyte reaction to OSCC. These results provide an immunobiological basis for the response of OSCC to CDDP and will contribute to our biological understanding of the action of novel combination therapy including immunotherapy together with platinum-based chemotherapy for OSCC.

Abstract 3004: Novel effect of Selumetinib-mediated autophagy via HSF1 in K-Ras mutant pancreatic cancer

Abstract Introduction: K-Ras mutant cancers such as pancreatic cancer are a major cause of cancer-related death and are difficult to treat, with a five-year survival rate of less than 6%. It is reported that oncogenic K-Ras signaling passes through RAF/MEK/ERK pathways. Selumetinib (AZD6244) is a selective MEK inhibitor for K-Ras mutant cancers dependent on mitogen-activated protein kinase (MAPK) signaling pathway and is currently in phase II trials. However, the underlying mechanisms of action are not well known. Autophagy is a self-digest pathway to degrade cellular organelles and macromolecules in maintaining proteome homeostasis, and it is suppressed by the mammalian target of rapamycin complex 1 (mTORC1), a master regulator of translation and autophagy. mTOR activity is known to be activated by AMP-activated protein kinase (AMPK) and negatively regulated by c-Jun N-terminal kinase 1/2 (JNK1/2) MAPK signaling. Autophagy is thought to play protective and suppressive roles in cancer; however, the molecular basis for the relationship between the induction of autophagy and the initiation of pancreatic malignancy is currently unknown. Heat shock factor 1 (HSF1), a key transcription factor involved in proteotoxic stress response via protein-folding. HSF1 plays a pro-oncogenic role in the development of cancer by regulating signaling transduction and translation during tumorigenesis. HSF1 is reportedly hyperactive in pancreatic cancers. AZD6244 reportedly decreases HSF1 phosphorylation at Ser326 and HSF1 expression in melanomas and therefore abates cancer development. However, the detailed mechanism by which HSF1 engages in AZD6244-mediated autophagy in K-Ras mutant human pancreatic cancer is not fully understood. Objectives: This study aims to investigate the role of HSF1 in AZD6244-mediated autophagy in K-Ras mutant human pancreatic cancer. Methods and Results: AZD6244 induces robust autophagy response in human pancreatic cells with hyperactive K-Ras signaling. Simultaneously, AZD6244 reduces HSF1 phosphorylation at Ser326 and HSF1 expression in human pancreatic cancer cells. Intriguingly, genetic deletion of HSF1 induces autophagy under starvation condition. In addition, AZD6244 induces phosphorylation of AMPK Ser172 and JNK1/2 T183/Y185 and decreases mTORC1 activity in human pancreatic cancer cells. Pharmaceutical inhibition of AMPK or genetic deletion of JNK1/2 prevents AZD6244-induced autophagy. Furthermore, HSF1 knockdown induces phosphorylation of Unc-51-like kinase 1(ULK1) Ser555 and decreases ULK Ser757, which is mediated by AMPK and mTOR, respectively. Conclusion: HSF1 plays an integral role in Selumetinib-mediated autophagy through AMPK and/or JNK1/2 signaling in K-Ras mutant human pancreatic cancer cells. Citation Format: Shruti Ghai, Alex Young, Kuo-Hui Su. Novel effect of Selumetinib-mediated autophagy via HSF1 in K-Ras mutant pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3004.

Propionate and Dietary Fermentable Fibers Upregulate Intestinal Heat Shock protein70 in Intestinal Caco-2 Cells and Mouse Colon.

Short-chain fatty acids (SCFAs), including propionate, are major metabolites of intestinal microorganisms and play an essential role in regulating intestinal epithelial integrity. Heat shock proteins (HSPs) promote cellular homeostasis under physiological and stressed conditions. This study aimed to investigate the regulation of intestinal HSP70 by propionate in human intestinal Caco-2 cells and the colon of fermentable dietary fiber (DF)-fed mice and germ-free mice. The results showed that propionate increased Hspa1a (HSP70 mRNA) level in Caco-2 cells, upregulated HSP70 protein, and phosphorylation of heat shock factor 1; however, the latter two were reduced by mitogen-activated protein kinases and the mechanistic target of rapamycin inhibitors. Feeding fermentable DFs, such as guar gum (GG) and partially hydrolyzed GG, increased both cecal SCFAs and colonic HSP70 expression, both of which were reduced in germ-free mice than in specific-pathogen-free mice. Collectively, the propionate-induced HSP70 expression was shown to be possibly involved in intestinal homeostasis.

The Novel Protein HSF1 Stress Pathway Inhibitor Bisamide CCT361814 Demonstrates Pre-Clinical Anti-Tumor Activity in Myeloma

IntroductionMultiple myeloma (MM) is a plasma cell malignancy, characterized by the constant production of immunoglobulins (Ig), making the tumor cells reliant on protein homeostasis for their survival. MM cells are therefore highly susceptible to therapies that target protein homeostasis, such as proteasome inhibitors. Several protein homeostasis mechanisms, when disturbed, converge on the heat shock pathway, a critical survival pathway for tumor cells comprising among other components molecular chaperones, the heat shock proteins (HSPs), and heat shock factor 1 (HSF1) - a master transcription factor and regulator of the heat shock response (HSR). Targeting HSP90 with small-molecule inhibitors has anti-myeloma activity (Stuhmer et al, 2009; Ishii et al, 2011), but leads to compensatory activation of the HSR, which makes inhibition of the HSR itself an attractive target. Recently, we discovered a chemical probe bisamide (CCT251236) that disrupts the HSF1 pathway (Cheeseman MD et al, 2017). This led to the identification of CCT361814 with bioavailability features compatible with clinical use. Here we present first data demonstrating marked antitumor activity by CCT361814 in MM.Methods and ResultsWe evaluated CCT361814 activity in 16 human myeloma cell line (HMCL) models including KMS11, KMS12BM, H929, L363 and RPMI-8226 using MTS cell proliferation assay and found that most HMCL cell lines displayed a GI50 in the nanomolar range (20-300nM, median GI50 147nM) whilst bone marrow stromal HS5 cells had a GI50 of around 700nM suggesting a therapeutic window. Cell lines with the t(11;14) translocation affecting CCND1 such as MOLP8, U266 and KMS12BM appeared to be particularly sensitive to the drug with median GI50 values of 88nM. Western blot analysis showed that CCT361814 caused a concentration-dependent loss of HSP27 and HSP72, as markers of HSF-1 pathway inhibition, and a reduced HSF1 phosphorylation at serine 326. Annexin V/DAPI staining was performed in 8 cell lines and demonstrated consistent induction of apoptosis in all lines, with MM1.S as an example showing a 9-fold increase in apoptosis compared to vehicle control (student's t-test, P=<0.0001). Increased autophagy was observed in L363 and RPMI-8226 cells as assessed by flow cytometry. This was also accompanied by increased cleaved caspase 3 and LC3 I-II conversion as detected by western blotting. Gene expression profiling was performed using Affymetrix U133 plus 2 arrays in L363, RPMI-8226 and KMS11 to observe any differentially expressed genes. Data were normalised using robust multi-array average (RMA) and analysed for differential gene expression using the limma package in R, employing a 2.5-fold change cut-off and significant false discovery rate adjusted p-values (P=<0.05). Overrepresentation enrichment analysis identified several upregulated genes belonging to the nucleic acid binding transcription factor activity molecular function including genes such as CEBP (P=0.02), DDIT3 (P=0.01), ATF3 (P=0.04) in all MM lines studied. DDIT3 and ATF3 along with other UPR markers such as XBP-1 splicing were also assayed by qRT-PCR and showed a significant upregulation in a dose-dependent manner in L363, KMS12BM, KMS11, RPMI-8226 and H929 cells (P=<0.05 for all). The analysis also revealed significantly downregulated genes belonging to chaperone binding group (P=0.024) and unfolded protein binding group (P=0.05) and genes encoding heat shock proteins such as HSPA8 (P=0.00011), HSPA1A (P=0.001) and HSP4L (P=0.03), consistent with functional work showing that CCT361814 affects the heat shock response.ConclusionsOur data shows that CCT361814 affects the heat shock pathway as evidenced by a concentration-dependent loss of HSP27 and HSP72 proteins and also has highly potent anti-myeloma activity in all HMCL models studied. Further functional validation of the gene expression data and assessing the effect of CCT361814 treatment in combination with clinically used drugs is underway. Our discovery of CCT361814 and its potent inhibition of the HSR in protein homeostasis-addicted MM cells makes it an attractive drug to be further evaluated in early phase clinical trials. DisclosuresWorkman:Institute of Cancer Research: Employment. Jones:Institute of Cancer Research: Employment. Kaiser:Celgene: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Other: Travel expenses; Amgen: Consultancy, Honoraria; Takeda: Consultancy; Janssen: Honoraria; Chugai: Consultancy.

Inhibition of Translation Initiation and Heat Shock Factor 1 (HSF1): A Novel Strategy Targeting FLT3 Double Mutant and Non-FLT3 Mutant AML Progenitor/Stem Cells

The transcription factor Heat shock factor 1 (HSF1) is a key sensor of proteotoxic stress and plays a major role in cancer biology. In various tumors, downregulation of HSF1 inhibits growth and induces cell death, and its activation was reported to be an adverse prognostic factor in breast and lung cancers. We have reported that inhibition of translation represses DNA-binding of HSF1 in cancers; large scale drug screening identified the eIF4a antagonist rohinitib (RHT) as a potent inhibitor of the HSF1 activation state (Santagata et al, Science 2013). Review of public databases provided us with additional rationale for targeting HSF1 and eIF4a in acute myeloid leukemia (AML): 1) mRNA levels of HSPA8, a primary HSF1 target, are correlated with poor prognosis in AML; 2) eIF4a mRNA levels are highest in AML among 12 cancer types; and 3) Gene set enrichment analysis using microarray dataset of functionally-defined leukemia stem cells (LSCs) and non-LSCs (Nature, Ng et al), based on intra-patient comparisons, reveals that a gene set for translation initiation is highly enriched in LSCs (NES 2.73, FDR = 0.000, p= 0.000).RHT induced marked apoptogenic effects in seven human AML cell lines in culture at low nanomolar concentrations (LD50s; 9.5 to 99.5 nM, IC50s; 4.7 to 8.8 nM, based on AnnexinV/PI-positivity at 72hr). RHT inhibited HSF1 transcriptional activity, as shown by 70% reduction in HSPA8 mRNA levels, and 50% reduction in a luciferase reporter driven by the HSPA1A or HSPA6 promoters. We then tested primary samples from 17 AML patients and bone marrow (BM) samples from eight healthy donors. RHT potently induced apoptosis in AML cells, while relatively sparing normal BM cells (%apoptosis: 65.4% vs 6.67%, P < 0.0001). Importantly, a similarly significant difference in sensitivity was also observed between AML and normal stem/progenitor cell populations (CD45+CD34+CD38-). We further found that the activity of RHT was significantly higher in FLT3-ITD than in FLT3-wt AMLs. Comparing isogenic versions of the cell line Ba/F3, RHT more potently killed FLT3-ITD cells than wild-type (wt) cells (LD50s; 65.3 vs 20.1 nM). Immunoblot analysis showed higher activation-associated phospho-HSF1 (Serine 326) in FLT3-ITD Ba/F3 than in FLT3-wt cells, and growth inhibition by HSF1 knockdown was FLT3-ITD-dependent in those lines, indicating greater dependence of FLT3-ITD cells on HSF1. In an AML xenograft model with GFP-luciferase labeled MOLM-13 cells, RHT significantly reduced tumor burden as measured by bioluminescence imaging and reduction in GFP+ leukemic cells peripheral blood (PB) and BM by day 16. Median survival of the treatment group was significantly longer (24 vs 18 days, p < 0.0001).We also found that RHT was synergistic when combined with FLT3 inhibitors of Type I (sorafenib, quizartinib, midostaurin) or Type II (gilteritinib, and E-6201). Immunoblot analysis showed that oncogenic FLT3 activation is associated with increased phosphorylation of HSF1 (S326), suggesting that decreased phosphorylation of the S326 site associated with FLT3 inhibition and diminished DNA-binding by HSF1 driven by eIF4a inhibition combine to promote synergistic lethality. In support of this, HSF1 knockdown sensitized MOLM-13 and FLT3-ITD+ Ba/F3 cells to FLT3 inhibitors. Importantly, similar combinatorial synergistic effects with type II FLT3 inhibitors were observed in cells with dual mutations of FLT3-ITD and FLT3-D835Y/H. This observation has significant clinical relevance because the development of secondary point mutations is a major resistance mechanism against FLT3 inhibitors.Finally, to test the hypothesis that eIF4a inhibition impairs LSC functions, we treated primary patient-derived xenograft AML PDX cells with RHT ex vivo, then injected identical numbers of live cells into recipient NSG mice. Subsequent leukemia engraftment was delayed by one week vs. controls, and human CD45+ cells in PB and BM at 4 weeks post transplantation were significantly reduced (n = 9, 3.30% vs 0.26% in PB, P < 0.0001). This reduced engraftment capacity of RHT-treated PDX AML-LSCs indicates a toxic effect of eIF4a inhibition on AML-LSCs.Collectively, these preclinical findings strongly support the further development of eIF4a inhibitors for the treatment of AML, especially for high-risk FLT3-mutated AML, with additional potential as LSC-targeted therapy. DisclosuresNo relevant conflicts of interest to declare.

Curcumin increases heat shock protein 70 expression via different signaling pathways in intestinal epithelial cells

Intestinal inflammation is associated with the integrity of the intestinal epithelium, which forms a physical barrier against noxious luminal substances. Heat shock 70 kDa protein 1A (HSP70), a molecular chaperon that exerts a cytoprotective effect, regulates intestinal integrity. This study investigated the modulation of HSP70 expression by dietary polyphenols, with particular reference to curcumin, in human intestinal Caco-2 cells. Immunoblot analysis demonstrated that among the 21 different polyphenols tested, curcumin most potently increased HSP70 levels in Caco-2 cells without affecting cell viability. Curcumin also increased the phosphorylation of heat shock factor 1 (HSF1), a well-known transcription factor of HSP70. Promoter and qRT-PCR assays indicated that curcumin upregulated Hspa1a levels via transcriptional activation. Pharmacological inhibition of MEK, a mechanistic target of rapamycin, p38 mitogen-activated protein kinase, and phosphatidyl 3-inositol kinase suppressed curcumin-mediated HSP70 expression, whereas HSF1 phosphorylation was sensitive only to MEK inhibition. Taken together, curcumin increases the expression of HSP70 in intestinal Caco-2 cells via transcriptional activation, possibly enhancing cell integrity. The effects exerted by curcumin are regulated by various signaling pathways. Our findings will expectedly contribute to a deeper understanding of the regulation of intestinal HSP70 by dietary components.

Augmentation of the heat shock axis during exceptional longevity in Ames dwarf mice

How the heat shock axis, repair pathways, and proteostasis impact the rate of aging is not fully understood. Recent reports indicate that normal aging leads to a 50% change in several regulatory elements of the heat shock axis. Most notably is the age-dependent enhancement of inhibitory signals associated with accumulated heat shock proteins and hyper-acetylation associated with marked attenuation of heat shock factor 1 (HSF1)–DNA binding activity. Because exceptional longevity is associated with increased resistance to stress, this study evaluated regulatory check points of the heat shock axis in liver extracts from 12 months and 24 months long-lived Ames dwarf mice and compared these findings with aging wild-type mice. This analysis showed that 12M dwarf and wild-type mice have comparable stress responses, whereas old dwarf mice, unlike old wild-type mice, preserve and enhance activating elements of the heat shock axis. Old dwarf mice thwart negative regulation of the heat shock axis typically observed in usual aging such as noted in HSF1 phosphorylation at Ser307 residue, acetylation within its DNA binding domain, and reduction in proteins that attenuate HSF1–DNA binding. Unlike usual aging, dwarf HSF1 protein and mRNA levels increase with age and further enhance by stress. Together these observations suggest that exceptional longevity is associated with compensatory and enhanced HSF1 regulation as an adaptation to age-dependent forces that otherwise downregulate the heat shock axis.

Heat shock factor 1 (HSF1-pSer326) predicts response to bortezomib-containing chemotherapy in pediatric AML: a COG report

AbstractBortezomib (BTZ) was recently evaluated in a randomized phase 3 clinical trial by the Children's Oncology Group (COG) that compared standard chemotherapy (cytarabine, daunorubicin, and etoposide [ADE]) vs standard therapy with BTZ (ADEB) for de novo pediatric acute myeloid leukemia (AML). Although the study concluded that BTZ did not improve outcome overall, we examined patient subgroups benefiting from BTZ-containing chemotherapy using proteomic analyses. The proteasome inhibitor BTZ disrupts protein homeostasis and activates cytoprotective heat shock responses. Total heat shock factor 1 (HSF1) and phosphorylated HSF1 (HSF1-pSer326) were measured in leukemic cells from 483 pediatric patients using reverse phase protein arrays. HSF1-pSer326 phosphorylation was significantly lower in pediatric AML compared with CD34+ nonmalignant cells. We identified a strong correlation between HSF1-pSer326 expression and BTZ sensitivity. BTZ significantly improved outcome of patients with low-HSF1-pSer326 with a 5-year event-free survival of 44% (ADE) vs 67% for low-HSF1-pSer326 treated with ADEB (P = .019). To determine the effect of HSF1 expression on BTZ potency in vitro, cell viability with HSF1 gene variants that mimicked phosphorylated (S326A) and nonphosphorylated (S326E) HSF1-pSer326 were examined. Those with increased HSF1 phosphorylation showed clear resistance to BTZ vs those with wild-type or reduced HSF1-phosphorylation. We hypothesize that HSF1-pSer326 expression could identify patients who benefit from BTZ-containing chemotherapy.

Thrombospondin 4/integrin \u03b12/HSF1 axis promotes proliferation and cancer stem-like traits of gallbladder cancer by enhancing reciprocal crosstalk between cancer-associated fibroblasts and tumor cells

BackgroundCancer-associated fibroblasts (CAFs), the primary component of tumor stroma in tumor microenvironments, are well-known contributors to the malignant progression of gallbladder cancer (GBC). Thrombospondins (THBSs or TSPs) comprise a family of five adhesive glycoproteins that are overexpressed in many types of cancers. However, the expression and potential roles of TSPs in the crosstalk between CAFs and GBC cells has remained unclear.MethodsPeritumoral fibroblasts (PTFs) and CAFs were extracted from GBC tissues. Thrombospondin expression in GBC was screened by RT-qPCR. MTT viability assay, colony formation, EdU incorporation assay, flow cytometry analysis, Transwell assay, tumorsphere formation and western blot assays were performed to investigate the effects of CAF-derived TSP-4 on GBC cell proliferation, EMT and cancer stem-like features. Subcutaneous tumor formation models were established by co-implanting CAFs and GBC cells or GBC cells overexpressing heat shock factor 1 (HSF1) to evaluate the roles of TSP-4 and HSF1 in vivo. To characterize the mechanism by which TSP-4 is involved in the crosstalk between CAFs and GBC cells, the levels of a variety of signaling molecules were detected by coimmunoprecipitation, immunofluorescence staining, and ELISA assays.ResultsIn the present study, we showed that TSP-4, as the stromal glycoprotein, is highly expressed in CAFs from GBC and that CAF-derived TSP-4 induces the proliferation, EMT and cancer stem-like features of GBC cells. Mechanistically, CAF-secreted TSP-4 binds to the transmembrane receptor integrin α2 on GBC cells to induce the phosphorylation of HSF1 at S326 and maintain the malignant phenotypes of GBC cells. Moreover, the TSP-4/integrin α2 axis-induced phosphorylation of HSF1 at S326 is mediated by Akt activation (p-Akt at S473) in GBC cells. In addition, activated HSF1 signaling increased the expression and paracrine signaling of TGF-β1 to induce the transdifferentiation of PTFs into CAFs, leading to their recruitment into GBC and increased TSP-4 expression in CAFs, thereby forming a positive feedback loop to drive the malignant progression of GBC.ConclusionsOur data indicate that a complex TSP-4/integrin α2/HSF1/TGF-β cascade mediates reciprocal interactions between GBC cells and CAFs, providing a promising therapeutic target for gallbladder cancer patients.

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.

17β-Estradiol Activates HSF1 via MAPK Signaling in ERα-Positive Breast Cancer Cells.

Heat Shock Factor 1 (HSF1) is a key regulator of gene expression during acute environmental stress that enables the cell survival, which is also involved in different cancer-related processes. A high level of HSF1 in estrogen receptor (ER)-positive breast cancer patients correlated with a worse prognosis. Here we demonstrated that 17β-estradiol (E2), as well as xenoestrogen bisphenol A and ERα agonist propyl pyrazole triol, led to HSF1 phosphorylation on S326 in ERα positive but not in ERα-negative mammary breast cancer cells. Furthermore, we showed that MAPK signaling (via MEK1/2) but not mTOR signaling was involved in E2/ERα-dependent activation of HSF1. E2­activated HSF1 was transcriptionally potent and several genes essential for breast cancer cells growth and/or ERα action, including HSPB8, LHX4, PRKCE, WWC1, and GREB1, were activated by E2 in a HSF1-dependent manner. Our findings suggest a hypothetical positive feedback loop between E2/ERα and HSF1 signaling, which may support the growth of estrogen-dependent tumors.

HSF1 phosphorylation by cyclosporin A confers hyperthermia sensitivity through suppression of HSP expression

Heat shock factor 1 (HSF1) is a transcription factor essential for tumorigenesis, and targeting HSF1 may be effective in combined therapeutics for cervical cancer. Cyclosporin A (CsA) is an immunosuppressant that has revolutionized organ transplantation. However, the roles and regulatory mechanisms by which CsA modulates HSP expression remain largely unknown. In this study, we found that CsA pretreatment prevented induction of HSPs during heat shock by enhancing the phosphorylation of Ser303 and Ser307 on HSF1 and thus inhibiting its transcriptional activity. Suppression of ERK1/2, GSK3β and CK2 activities attenuated CsA-induced down-regulation of HSP expression and up-regulation of HSF1 phosphorylation. CsA interfered with HSF1-SSBP1 complex formation and HSF1 nuclear translocation and recruitment to the HSP70 promoter. CsA clearly caused HeLa cell death during proteotoxic stress through reduced expression of HSPs. These results indicate that CsA suppresses HSP induction during heat shock by regulating the phosphorylation and nuclear translocation of HSF1. Our results provide a conceptual framework for the development of novel therapeutic strategies for cervical cancer through application of CsA during hyperthermia or chemotherapy.

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.