Heat Shock Factor 1 Expression Research Articles

Heat shock factor 1 inhibition enhances the effects of modulated electro hyperthermia in a triple negative breast cancer mouse model

Female breast cancer is the most diagnosed cancer worldwide. Triple negative breast cancer (TNBC) is the most aggressive type and there is no existing endocrine or targeted therapy. Modulated electro-hyperthermia (mEHT) is a non-invasive complementary cancer therapy using an electromagnetic field generated by amplitude modulated 13.56 MHz frequency that induces tumor cell destruction. However, we have demonstrated a strong induction of the heat shock response (HSR) by mEHT, which can result in thermotolerance. We hypothesized that inhibition of the heat shock factor 1 (HSF1) can synergize with mEHT and enhance tumor cell-killing. Thus, we either knocked down the HSF1 gene with a CRISPR/Cas9 lentiviral construct or inhibited HSF1 with a specific small molecule inhibitor: KRIBB11 in vivo. Wild type or HSF1-knockdown 4T1 TNBC cells were inoculated into the mammary gland’s fat pad of BALB/c mice. Four mEHT treatments were performed every second day and the tumor growth was followed by ultrasound and caliper. KRIBB11 was administrated intraperitoneally at 50 mg/kg daily for 8 days. HSF1 and Hsp70 expression were assessed. HSF1 knockdown sensitized transduced cancer cells to mEHT and reduced tumor growth. HSF1 mRNA expression was significantly reduced in the KO group when compared to the empty vector group, and consequently mEHT-induced Hsp70 mRNA upregulation diminished in the KO group. Immunohistochemistry (IHC) confirmed the inhibition of Hsp70 upregulation in mEHT HSF1-KO group. Demonstrating the translational potential of HSF1 inhibition, combined therapy of mEHT with KRIBB11 significantly reduced tumor mass compared to either monotherapy. Inhibition of Hsp70 upregulation by mEHT was also supported by qPCR and IHC. In conclusion, we suggest that mEHT-therapy combined with HSF1 inhibition can be a possible new strategy of TNBC treatment with great translational potential.

Novel mechanism of drug resistance triggered by tumor-associated macrophages through Heat Shock Factor-1 activation

Macrophages constitute a major part of tumor microenvironment, and most of existing data demonstrate their ruling role in the development of anti-drug resistance of cancer cell. One of the most powerful protection system is based on heat shock proteins whose synthesis is triggered by activated Heat Shock Factor-1 (HSF1); the inhibition of the HSF1 with CL-43 sensitized A549 lung cancer cells to the anti-cancer effect of etoposide. Notably, analyzing A549 tumor xenografts in mice we observed nest-like pattern of co-localization of A549 cells demonstrating enhanced expression of HSF1 with macrophages, and decided to check whether the above arrangement has a functional value for both cell types. It was found that the incubation of A549 or DLD1 colon cancer cells with either human monocytes or THP1 monocyte-like cells activated HSF1 and increased resistance to etoposide. Importantly, the same effect was shown when primary cultures of colon tumors were incubated with THP1 cells or with human monocytes. To prove that HSF1 is implicated in enhanced resistance caused by monocytic cells, we generated an A549 cell subline devoid of HSF1 which did not respond to incubation with THP1 cells. The pharmacological inhibition of HSF1 with CL-43 also abolished the effect of THP1 cells on primary tumor cells, highlighting a new target of tumor-associated macrophages in a cell proteostasis mechanism.

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.

Mechanically sensitive HSF1 is a key regulator of left-right symmetry breaking in zebrafish embryos

The left-right symmetry breaking of vertebrate embryos requires nodal flow. However, the molecular mechanisms that mediate the asymmetric gene expression regulation under nodal flow remain elusive. Here, we report that heat shock factor 1 (HSF1) is asymmetrically activated in the Kupffer's vesicle of zebrafish embryos in the presence of nodal flow. Deficiency in HSF1 expression caused a significant situs inversus and disrupted gene expression asymmetry of nodal signaling proteins in zebrafish embryos. Further studies demonstrated that HSF1 is a mechanosensitive protein. The mechanical sensation ability of HSF1 is conserved in a variety of mechanical stimuli in different cell types. Moreover, cilia and Ca2+-Akt signaling axis are essential for the activation of HSF1 under mechanical stress invitro and invivo. Considering the conserved expression of HSF1 in organisms, these findings unveil a fundamental mechanism of gene expression regulation by mechanical clues during embryonic development and other physiological and pathological transformations.

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.

HSF1 activates the FOXO3a-ΔNp63α-CDK4 axis to promote head and neck squamous cell carcinoma cell proliferation and tumour growth.

Head and neck squamous cell carcinoma (HNSCC) is one of the most prevalent cancers worldwide. Heat shock factor 1 (HSF1) is a conserved transcriptional factor that plays a critical role in maintaining cellular proteostasis. However, the role of HSF1 in HNSCC development remains largely unclear. Here, we report that HSF1 promotes forkhead box protein O3a (FOXO3a)-dependent transcription of ΔNp63α (p63 isoform in the p53 family; inhibits cell migration, invasion, and metastasis), which leads to upregulation of cyclin-dependent kinase 4 expression and HNSCC tumour growth. Ablation of HSF1 or treatment with KRIBB11, a specific pharmacological inhibitor of HSF1, significantly suppresses ΔNp63α expression and HNSCC tumour growth. Clinically, the expression of HSF1 is positively correlated with the expression of ΔNp63α in HNSCC tumours. Together, this study demonstrates that the HSF1-ΔNp63α pathway is critically important for HNSCC tumour growth.

HSF1 Alleviates Brain Injury by Inhibiting NLRP3-Induced Pyroptosis in a Sepsis Model.

Sepsis, which could cause a systemic inflammatory response, is a life-threatening disease with a high morbidity and mortality rate. There is evidence that brain injury may be related to severe systemic infection induced by sepsis. The brain injury caused by sepsis could increase the risk of mortality in septic patients, which seriously affects the septic patient's prognosis of survival. Although there remains a focus on sepsis research, clinical measures to prevent and treat brain injury in sepsis are not yet available, and the high mortality rate is still a big health burden. Therefore, it is necessary to investigate the new molecules or regulated pathways that can effectively inhibit the progress of sepsis. NLR family pyrin domain-containing 3 (NLRP3) increased in the procession of sepsis and functioned as the key regulator of pyroptosis. Heat shock factor 1 (HSF1) can protect organs from multiorgan dysfunction syndrome induced by lipopolysaccharides in mice, and NLRP3 could be inhibited by HSF1 in many organs. However, whether HSF1 regulated NLRP3 in sepsis-induced brain injury, as well as the detailed mechanism of HSF1 in brain injury, remains unknown in the sepsis model. In this research, we try to explore the relationship between HSF1 and NLRP3 in a sepsis model and try to reveal the mechanism of HSF1 inhibiting the process of brain injury. In this study, we used wild-type mice and hsf1 -/- mice for in vivo research and PC12 cells for in vitro research. Real-time PCR and Western blot were used to analyze the expression of HSF1, NLRP3, cytokines, and pyrolytic proteins. EthD-III staining was chosen to detect the pyroptosis of the hippocampus and PC12 cells. The results showed that HSF1 is negatively related to pyroptosis. The pyroptosis in cells of brain tissue was significantly increased in the hsf1 -/- mouse model compared to hsf1 +/+ mice. In PC12 cells, hsf1 siRNA can upregulate pyroptosis while HSF1-transfected plasmid could inhibit the pyroptosis. HSF1 could negatively regulate the NLRP3 pathway in PC12 cells, while hsf1 siRNA enhanced the pyroptosis in PC12 cells, which could be reversed by nlrp3 siRNA. These results imply that HSF1 could alleviate sepsis-induced brain injury by inhibiting pyroptosis through the NLRP3-dependent pathway in brain tissue and PC12 cells, suggesting HSF1 as a potential molecular target for treating brain injury in sepsis clinical studies.

HSF1 expression in tumor-associated macrophages promotes tumor cell proliferation and indicates poor prognosis in esophageal squamous cell carcinoma.

Tumor-associated macrophages (TAMs), are crucial for the survival and development of tumor cells. Heat shock factor 1 (HSF1) is a potent, complex carcinogenesis modulator, and esophageal cancer (EC) patients have a bad prognosis when HSF1 is highly expressed. HSF1's clinical importance and biological role in TAMs are still unknown. The HSF1 expression profile and patient survival information were analyzed from the TCGA database. The infiltration of different types of immune cells in EC was evaluated based on HSF1 gene expression by Sangerbox 3.0. Immunochemistry was employed to assess HSF1 protein expression in 134 individuals with esophageal squamous cell carcinoma (ESCC), proceeded by association with clinicopathological variables. The role of macrophage-driven HSF1 were observed using HSF1-knockdown THP1 cells. High level of HSF1 have a poorer prognosis in individuals with EC. The expressing level of HSF1 was positively related to infiltration of M2 macrophages (P < 0.05). The expression of HSF1 in macrophages was an independent factor for DFS (P = 0.002) and OS (P = 0.002) in ESCC cases. HSF1 was up-regulated in IL-4 stimulation THP1 cells in a time-dependent manner. Under the heat stimulation condition, THP1-derived macrophages were more sensitive than tumor cells. Compared to IL-4 induced-THP1 cells control, the HSF1 knockdown in THP1 cell inhibited the growth and proliferation of ESCC cells. The up-regulation of HSF1 was more rapid and could affect the proliferation of tumor cells in IL4-induced macrophages. The expression of HSF1 in TAMs can also serve as a marker for ESCC prognosis.

HSF1 is involved in immunotherapeutic response through regulating APOJ/STAT3-mediated PD-L1 expression in hepatocellular carcinoma

ABSTRACT Hepatocellular cancer (HCC) is a serious illness with high prevalence and mortality throughout the whole world. For advanced HCC, immunotherapy is somewhat impactful and encouraging. Nevertheless, a substantial proportion of patients with advanced HCC are still unable to achieve a durable response, owing to heterogeneity from clonal variability and differential expression of the PD-1/PD-L1 axis. Recently, heat shock factor 1 (HSF1) is recognized as an important component of tumor immunotherapeutic response as well as related to PD-L1 expression in cancer. However, the mechanism of HSF1 regulating PD-L1 in cancer, especially in HCC, is still not fully clear. In this study, we observed the significantly positive correlation between HSF1 expression and PD-L1 expression in HCC samples; meanwhile combination expressions of HSF1 and PD-L1 served as the signature for predicting prognosis of patients with HCC. Mechanistically, HSF1 upregulated PD-L1 expression by inducing APOJ expression and activating STAT3 signaling pathway in HCC. In addition, we explored further the potential values of targeting the HSF1-APOJ-STAT3 axis against CD8+ T cells-mediated cancer cells cytotoxicity. These findings unveiled the important involvement of HSF1 in regulating PD-L1 expression in HCC as well as provided a novel invention component for improving the clinical response rate and efficacy of PD-1/PD-L1 blockade.

Crosstalk between heat shock factor 1 and signal transducer and activator of transcription 3 mediated by interleukin-8 autocrine signaling maintains the cancer stem cell phenotype in liver cancer.

Liver cancer stem cells (LCSCs) cause therapeutic refractoriness and relapse in hepatocellular carcinoma. Heat shock factor 1 (HSF1) plays versatile roles in multiple cancers. However, the role of HSF1 in LCSCs is not well understood. This study investigated the function and signal mechanisms of HSF1 in maintaining LCSC phenotypes. We established two LCSC lines, HepG2-R and HuH-7-R. Constitutive activation of HSF1 was observed in these LCSCs. Specific short hairpin RNAs (shRNAs) and chemical inhibitors were used to identify the relationship between HSF1 expression and LCSCs phenotypes. We revealed a concomitant activation modality involving HSF1 and STAT3 in LCSCs and liver cancer tissues. We also found that liver cancer patients whose HSF1 and STAT3 mRNA expression levels were high presented with unfavorable clinicopathological characteristics. Moreover, the secretion of interleukin-8 (IL-8) was elevated in the LCSC medium and was directly regulated by HSF1 at the transcriptional level. In turn, IL-8 activated HSF1 and STAT3 signaling, and a neutralizing IL-8 antibody inhibited HSF1 and STAT3 activity, reduced cancer stem cell marker expression, and decreased LCSC microsphere formation. Simultaneous intervention with HSF1 and STAT3 led to synergistically suppressed stemness acquisition and growth suppression in the LCSCs in vivo and in vitro. Our study indicates that IL-8 mediates the crosstalk between the HSF1 and Stat3 signaling pathways in LCSCs and that the combined targeting of HSF1 and STAT3 is a promising treatment strategy for patients with advanced liver cancer.

Inhibition of Heat Shock Factor 1 Signaling Decreases Hepatoblastoma Growth via Induction of Apoptosis

Although rare compared with adult liver cancers, hepatoblastoma (HB) is the most common pediatric liver malignancy, and its incidence is increasing. Currently, the treatment includes surgical resection with or without chemotherapy, and in severe cases, liver transplantation in children. The effort to develop more targeted, HB-specific therapies has been stymied by the lack of fundamental knowledge about HB biology. Heat shock factor 1 (HSF1), a transcription factor, is a canonical inducer of heat shock proteins, which act as chaperone proteins to prevent or undo protein misfolding. Recent work has shown a role for HSF1 in cancer beyond the canonical heat shock response. The current study found increased HSF1 signaling in HB versus normal liver. It showed that less differentiated, more embryonic tumors had higher levels of HSF1 than more differentiated, more fetal-appearing tumors. Most strikingly, HSF1 expression levels correlated with mortality. This study used a mouse model of HB to test the effect of inhibiting HSF1 early in tumor development on cancer growth. HSF1 inhibition resulted in fewer and smaller tumors, suggesting HSF1 is needed for aggressive tumor growth. Moreover, HSF1 inhibition also increased apoptosis in tumor foci. These data suggest that HSF1 may be a viable pharmacologic target for HB treatment.

Clinical significance and potential mechanism of heat shock factor 1 in acute myeloid leukemia.

Background: Heat shock factor 1 (HSF1) is now considered to have the potential to be used as a prognostic biomarker in cancers. However, its clinical significance and potential function in acute myeloid leukemia (AML) remain underexplored.Methods: In this study, the expression pattern and clinical significance of HSF1 in AML were examined by integrating data from databases including The Cancer Genome Atlas (TCGA), The Genotype–Tissue Expression (GTEx), Vizome, Cancer Cell Line Encyclopedia (CCLE) and Gene Expression Omnibus (GEO). Linkedomics was applied to collect HSF1–related genes in AML. GeneMANIA was applied to outline HSF1–related functional networks. CancerSEA analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and Gene Set Enrichment Analysis (GSEA) were performed to mine the potential mechanism of HSF1 in leukemogenesis. Single–sample Gene Set Enrichment Analysis (ssGSEA) was applied to explore the correlation between HSF1 and infiltrating immune cells in AML.Results: HSF1 expression was elevated in AML compared to healthy controls and indicate a poor overall survival. HSF1 expression was significantly correlated with patients age, associated with patient survival in subgroup of bone marrow blasts (%) >20. Functional analyses indicated that HSF1 plays a role in the metastatic status of AML, and is involved in inflammation–related pathways and biological processes. HSF1 expression was significantly correlated with the immune infiltration of nature killer cells and T cell population.Conclusion: HSF1 plays a vital role in the molecular network of AML pathogenesis, and has the potential to be a biomarker for prognosis prediction.

Loss of heat shock factor 1 promotes hepatic stellate cell activation and drives liver fibrosis.

Liver fibrosis is an aberrant wound healing response that results from chronic injury and is mediated by hepatocellular death and activation of hepatic stellate cells (HSCs). While induction of oxidative stress is well established in fibrotic livers, there is limited information on stress‐mediated mechanisms of HSC activation. Cellular stress triggers an adaptive defense mechanism via master protein homeostasis regulator, heat shock factor 1 (HSF1), which induces heat shock proteins to respond to proteotoxic stress. Although the importance of HSF1 in restoring cellular homeostasis is well‐established, its potential role in liver fibrosis is unknown. Here, we show that HSF1 messenger RNA is induced in human cirrhotic and murine fibrotic livers. Hepatocytes exhibit nuclear HSF1, whereas stellate cells expressing alpha smooth muscle actin do not express nuclear HSF1 in human cirrhosis. Interestingly, despite nuclear HSF1, murine fibrotic livers did not show induction of HSF1 DNA binding activity compared with controls. HSF1‐deficient mice exhibit augmented HSC activation and fibrosis despite limited pro‐inflammatory cytokine response and display delayed fibrosis resolution. Stellate cell and hepatocyte‐specific HSF1 knockout mice exhibit higher induction of profibrogenic response, suggesting an important role for HSF1 in HSC activation and fibrosis. Stable expression of dominant negative HSF1 promotes fibrogenic activation of HSCs. Overactivation of HSF1 decreased phosphorylation of JNK and prevented HSC activation, supporting a protective role for HSF1. Our findings identify an unconventional role for HSF1 in liver fibrosis. Conclusion: Our results show that deficiency of HSF1 is associated with exacerbated HSC activation promoting liver fibrosis, whereas activation of HSF1 prevents profibrogenic HSC activation.

Melatonin attenuates MPP+-induced apoptosis via heat shock protein in a Parkinson's disease model

Heat shock proteins (HSPs) play an essential role as molecular chaperones to prevent abnormal protein aggregation and misfolding. Moreover, they protect dopamine neurons from oxidative stress, inflammation, and apoptosis, all well-known pathomechanisms of Parkinson's disease (PD). Melatonin is a potent antioxidant that has the beneficial ability to prevent neurodegenerative diseases like PD. We aimed to explore the protective properties of melatonin in an in vitro PD model, focusing on its underlying mechanism using HSPs. A 1-methyl-4-phenylpyridimium (MPP+)-induced toxin model was established with retinoic acid (RA)-differentiated SH-SY5Y cells. Cell viability and apoptosis were measured using MTT and DAPI. Intracellular reactive oxygen species (ROS) levels were measured by the cell-permeant fluorescent probe DCFH-DA. The level of malondialdehyde and the activities of superoxide dismutase and glutathione peroxidase were assessed using ELISA kits. Apoptotic markers of Bax, Bcl2, and cleaved caspase-3, as well as HSP70 and heat shock factor-1 (HSF1), were measured by Western blot. The melatonin effect through HSP70 was tested with silencing of HSF1 in the MPP + -treated SH-SY5Y cells. Melatonin can protect against MPP + -induced neuronal toxicity by promoting anti-oxidative and anti-apoptotic properties. SH-SY5Y cells exposed to melatonin with MPP + showed increased expression of HSP70 and HSF1 compared with those exposed to MPP + alone. However, siRNA-mediated downregulation of HSF1 significantly attenuated the protective effects of melatonin in the MPP + -induced in vitro PD model. Our findings revealed the protective roles of melatonin in an in vitro PD model. Melatonin can hinder the toxic effects of MPP + on dopaminergic neuronal cells via upregulation of the HSF1/HSP70 pathway. Further experimental studies would verify the therapeutic relevance of melatonin with HSP70 and HSF1 to prevent and decelerate PD-like neurodegeneration.

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.

Abstract ND08: NXP800: A first-in-class orally active, small-molecule HSF1 pathway inhibitor

Abstract We report the discovery of NXP800 — a first-in-class orally active HSF1 pathway inhibitor identified in our academic laboratory at the Institute of Cancer Research, London — and its progression into Phase I clinical trials in collaboration with our partner Nuvectis Pharma. HSF1 (Heat Shock Factor 1) is an ancient stress-inducible transcription factor that plays a key role in the transcriptional activation of the eukaryotic heat shock response. This leads to expression of many heat shock proteins, including molecular chaperones, and through this HSF1 acts as a master transcriptional regulator of proteostasis. Importantly, HSF1 is hijacked by cancer cells to activate a set of genes that overlaps with, but is not identical to, the classical heat shock response. The HSF1 pathway has been shown to play a key role in oncogenesis and the hallmark features of malignancy and is important in the initiation and progression of many experimental cancer models. Importantly, while HSF1 has been demonstrated to be important in mouse models of oncogenesis, knockout of the homologue of HSF1 has only modest effects in mice and flies under non-stressed laboratory conditions – supporting the potential for a therapeutic window. In addition, gene amplification, expression and activation of HSF1 has been shown to predict clinical outcome in many human cancers. Despite strong scientific rationale and pre-clinical validation, pharmaceutical exploitation has been limited because analysis of the available molecular structures of HSF1 shows it to be very difficult to drug. Because of this, we set out to discover inhibitors of the HSF1 pathway using an initial phenotypic screen that reports on the activation of the HSF1-regulated gene HSP72. This identified the bisamide series that was followed up by a detailed understanding of structure-activity relationships and medicinal chemistry optimization, particularly to improve pharmaceutical properties, leading to the selection of NXP800 (CCT361814) as the development candidate. NXP800 has good pharmacokinetic properties in mice, including oral bioavailability; also, pharmacodynamic studies show modulation of biomarkers of the HSF1 pathway and stress response in human tumour xenograft models. NXP800 exhibits striking therapeutic activity in xenografts of human ovarian clear cell ovarian cancer (OCCC) and endometrioid ovarian cancer, two serious conditions of high unmet medical need with limited treatment options – including prolonged tumour growth inhibition and regression at doses that show good toleration. The efficacy and tolerability data indicate a clear therapeutic window, supported by selectivity and safety profiting in pharmacology and kinome panels. In addition, we identified deficiency in ARID1A, a component of the SWI/SNF chromatin remodelling complex, as indicative of greater therapeutic responsiveness. This was confirmed through broader evaluation in human cancer cell line panels that also indicates therapeutic potential for NXP800 in additional cancer types. Studies to support mechanism of action and identify patient selection biomarkers will be described. A Phase 1 trial of NXP800 is now open, incorporating validated predictive, pharmacokinetic and pharmacodynamic biomarkers that provide a robust Pharmacological Audit Trail and support the prediction of dose-to-human. Citation Format: Paul Workman. NXP800: A first-in-class orally active, small-molecule HSF1 pathway inhibitor [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 ND08.

HSF1 Signaling Inhibits Apoptosis in Hepatoblastoma

IntroductionHepatoblastoma (HB) is the most common pediatric liver malignancy. While relatively rare, its incidence is increasing. Currently surgical resection with or without chemotherapy is the main treatment for the tumor though in severe cases children require liver transplantation. Development of precision therapy for the tumor has been hampered by gaps in fundamental knowledge about how the tumor develops and grows. Heat shock factor 1 (HSF1) is the master regulator of the heat shock response, which classically mediates cellular stress from heat but it likely has a cancer specific. Recent work has shown that HSF1 controls a suite of genes in cancer related to apoptosis, cell cycle signaling, DNA repair, energy metabolism and extracellular matrix formation. Previously, we conducted a meta‐analysis of six human HB transcriptomic data sets, which found expression levels of HSF1 was higher in tumors versus normal liver. Intriguingly, we found higher levels of HSF1 expression in tumors correlated with mortality. We wanted to investigate how inhibiting HSF1 impacted tumor growth in our lab’s mouse model of hepatoblastoma based on transfection with constitutively active forms of β‐catenin and yes‐associated protein 1 (Yap1). This model induces tumors that genetically and histologically resemble human hepatoblastoma. The objective of this study was to investigate the specific role of HSF1 signaling in the development of hepatoblastoma. Our hypothesis is inhibiting HSF1 in our mouse model of hepatoblastoma leads to increased apoptosis in tumor cells and thus decreased tumor size and number.MethodsWe inhibited HSF1 signaling in mice by co‐transfecting a dominant negative HSF1 (dnHSF1) plasmid along with β‐catenin and Yap1 plasmids. The dnHSF1 forms a truncated form of the protein that cannot initiate transcription of target genes. Mice injected with just β‐catenin and Yap1 plasmids served as controls. Eight weeks after transfection, the mice were harvested and the livers were preserved for analysis. Liver and body weights of each mouse was measured to calculate liver‐to‐body weight ratio, an index of tumor burden. At least three mice per condition was assessed.ResultsWe found transfection with the dnHSF1 lead to decreased liver‐to‐body weight ratio compared to control mice transfected with just the β‐catenin and Yap1 plasmids (p‐value&lt;0.05)(Figure 1). What few tumor foci remained featured larger, more differentiated cells. Indeed, these foci showed positive staining via immunohistochemistry (IHC) for both activated mTORC1 and glutamine synthetase, markers seen early in tumor development. Next, we assessed apoptosis by conducting IHC for cleaved caspase 3 and by performing a TUNEL assay on liver sections from both dnHSF1/β‐catenin/Yap1 mice and from control β‐catenin/Yap1 animals. Staining for both markers was increased nearly three times in dnHSF1 treated animals versus control(p‐value&lt;0.05)(Figure 2).ConclusionsWe conclude inhibiting HSF1 signaling in burgeoning hepatoblastoma cells leads to smaller, more differentiated tumors via increased apoptosis. More work is needed to determine if HSF1 is a viable pharmacologic target for hepatoblastoma.

Heat shock factor 1 inhibition sensitizes pancreatic cancer to gemcitabine via the suppression of cancer stem cell-like properties

Pancreatic cancer is a fatal disease with poor prognosis. Gemcitabine has been regarded as the mainstay of chemotherapy for pancreatic cancer; however, it is accompanied with a high rate of chemoresistance. Cancer stem cells (CSCs) are characterized by resistance to traditional chemo- and radiotherapies. We have previously reported that heat shock factor 1 (HSF1) is involved in the invasion and metastasis of pancreatic cancer, a highly conserved transcriptional factor that mediates the canonical proteotoxic stress response. Here, we investigate whether HSF1 contributes to the chemoresistance of pancreatic cancer cells caused by gemcitabine and explore the underlying mechanisms. Genetically engineered mice (LSL-KrasG12D/+; Trp53fl/+; Pdx1-Cre mice), which spontaneously develop pancreatic cancer, were used to examine the sensitivity of pancreatic cancer to gemcitabine in vivo. We found that HSF1 was enriched in sphere-forming cancer cells. Panc-1 and MiaPaCa-2 cells treated chronically with gemcitabine displayed increased transcription and expression of CSC-associated markers. In addition, gemcitabine-surviving Panc-1 and MiaPaCa-2 cells showed an increased ability to form tumorspheres. Moreover, we observed that gemcitabine treatment increased the activity and expression of HSF1, as well as transcription of its downstream targets. Finally, HSF1 inhibition significantly suppressed the expression of CSC-associated markers, augmented the cancer-killing property of gemcitabine, and increased chemosensitivity to gemcitabine in vivo. Our study reveals a novel mechanism in which HSF1 promotes the chemoresistance of pancreatic cancer to gemcitabine by modulating CSC-like properties. Targeting HSF1 could be thus a rational strategy to improve treatment outcomes.

Integrated bioinformatics and experiments reveal the roles and driving forces for HSF1 in colorectal cancer

ABSTRACT Heat shock factor 1 (HSF1) has watershed significance in different tumors. However, the roles and driving forces for HSF1 in colorectal cancer (CRC) are poorly understood. Our study integrally analyzed the roles and driving forces for HSF1 in CRC by bioinformatics and experiments. The expression and prognostic characteristics of HSF1 were analyzed via UALCAN, GEPIA2, TISIDB, Prognoscan and HPA databases. Then, we analyzed the correlation between HSF1 expression and immune features via TIMER2 database. Subsequently, we explored the driving forces for HSF1 abnormal expression in CRC by bioinformatics and experiments. Our results showed that HSF1 was overexpressed and correlated with poor prognosis in CRC. And the expression of HSF1 was significantly correlated with multiple immune cell infiltration and was negatively correlated with immunomodulators such as programmed cell death 1 ligand 1(PD-L1). Along with many driver genes in particular TP53, super-enhancer, miRNA and DNA methylation were all responsible for HSF1 overexpression in CRC. Moreover, we demonstrated that β-catenin could promote the translation process of HSF1 mRNA by interacting with HuR, which could directly bind to the coding sequence (CDS) region of HSF1 mRNA. Collectively, HSF1 may be useful as a diagnostic and prognostic biomarker for CRC. HSF1 was closely correlated with immune features. Genetic and epigenetic alterations contributed to HSF1 overexpression in CRC. More importantly, we demonstrated that HSF1 may be regulated at the level of mRNA translation by β-catenin-induced HuR activity.

Heat shock factor 1 in brain tumors: a link with transient receptor potential channels TRPV1 and TRPA1.

Novel data report a "cross-talk" between Heat-Shock Factor 1 (HSF1) and the transient receptor potential vanilloid 1 cation channel (TRPV1) located in the cell membrane, introducing these channels as possible drug targets for the regulation of HSF1 activation. This study aims to investigate the co-expression of TRPV1 and HSF1 in human brain tumors. Additionally, the expression of the transient receptor potential ankyrin 1 channel (TRPA1), which is co-operated with TRPV1 in a plethora of cells, was studied. Immunohistochemical staining for HSF1, TRPV1 and TRPA1 expression was quantitatively analyzed in paraffin-embedded semi-serial tissue sections from 74 gliomas and 71 meningiomas. mRNA levels of HSF1, TRPV1 and TRPA1 were evaluated using real-time PCR. Although HSF1 was significantly increased compared with TRPV1/TRPA1 (p ≤ 0.001) in both gliomas and meningiomas, high co-expression levels for HSF1, TRPV1 and TRPA1 were found in 62.50% of diffuse fibrillary astrocytomas (WHO, grade II), 37.50% of anaplastic astrocytomas (WHO, grade III), 16.32% of glioblastomas multiforme (WHO, grade IV), and 42.25% of meningiomas (WHO, grade I and II). Correlation analysis revealed a relationship of HSF1 with TRPV1/TRPA1 in diffuse fibrillary astrocytomas (WHO, grade II) and benign meningiomas (WHO, grade I) contrary to glioblastomas multiforme (WHO, grade IV) and high grade meningiomas (WHO, grade II). Importantly, TRPA1 and TRPV1 expression levels were significantly increased in meningiomas compared with astrocytic tumors (p < 0.05). In conclusion, HSF1 and TRPV1/TRPA1 co-expression may be implicated in the pathogenesis of human brain tumors and should be considered for the therapeutic approaches for these tumors.