Extracellular Heat Shock Protein Research Articles

Extracellular Hsp90 of Candida albicans contributes to the virulence of the pathogen by activating the NF-κB signaling pathway and inducing macrophage pyroptosis

Strategies aimed at targeting fungal extracellular heat shock protein 90 (eHsp90) using vaccines and antibodies have demonstrated encouraging potential in the prevention and management of invasive fungal diseases (IFDs). However, the precise underlying mechanism by which eHsp90 contributes to the heightened virulence of Candida albicans (C. albicans) remains an enigma, awaiting further elucidation. In our current research, we have found that the 47-kDa fragment of C. albicans Hsp90 (CaHsp90), which serves as the primary antigenic determinant, is not degraded within C. albicans cells. Moreover, we have discovered that extracellular CaHsp90 (eCaHsp90) is derived from the components of lysed C. albicans cells. We also generated recombinant CaHsp90 in Escherichia coli, and found that eCaHsp90 spreads beyond the initial C. albicans colonization site, thereby enhancing the overall virulence of the organism. Our results further clarify that eCaHsp90 activates the nuclear factor kappa-B (NF-κB) signaling pathway and upregulates the expression of NACHT, LRR, and PYD domains-containing protein 3 (NLRP3). This upregulation results in the activation of Gasdermin D (GSDMD) and subsequent macrophage pyroptosis, ultimately increasing the virulence of C. albicans. This study provides valuable insights into the mechanism by which eCaHsp90 contributes to the virulence of C. albicans, offering a pharmacological basis for antifungal strategies targeting fungal eHsp90.

1G6-D7 Inhibits Homologous Recombination Repair by Targeting Extracellular HSP90α to Promote Apoptosis in Non-Small Cell Lung Cancer.

Despite recent advances in treatment, non-small cell lung cancer (NSCLC) continues to have a high mortality rate. Currently, NSCLC pathogenesis requires further investigation, and therapeutic drugs are still under development. Homologous recombination repair (HRR) repairs severe DNA double-strand breaks. Homologous recombination repair deficiency (HRD) occurs when HRR is impaired and causes irreparable double-strand DNA damage, leading to genomic instability and increasing the risk of cancer development. Poly(ADP-ribose) polymerase (PARP) inhibitors can effectively treat HRD-positive tumors. Extracellular heat shock protein 90α (eHSP90α) is highly expressed in hypoxic environments and inhibits apoptosis, thereby increasing cellular tolerance. Here, we investigated the relationship between eHSP90α and HRR in NSCLC. DNA damage models were established in NSCLC cell lines (A549 and H1299). The activation of DNA damage and HRR markers, apoptosis, proliferation, and migration were investigated. In vivo tumor models were established using BALB/c nude mice and A549 cells. We found that human recombinant HSP90α stimulation further activated HRR and reduced DNA damage extent; however, eHSP90α monoclonal antibody, 1G6-D7, effectively inhibited HRR. HRR inhibition and increased apoptosis were observed after LRP1 knockdown; this effect could not be reversed with hrHSP90α addition. The combined use of 1G6-D7 and olaparib caused significant apoptosis and HRR inhibition in vitro and demonstrated promising anti-tumor effects in vivo. Extracellular HSP90α may be involved in HRR in NSCLC through LRP1. The combined use of 1G6-D7 and PARP inhibitors may exert anti-tumor effects by inhibiting DNA repair and further inducing apoptosis of NSCLC cells.

Abstract 3353: The emerging role of extracellular HSP90 in prostate cancer progression to a metastatic phenotype

Abstract Background: Metastasis is the central cause of lethal prostate cancer (PCa). Mechanisms regulating metastasis represent high-potential therapeutic targets for preventing development of lethal PCa. Inhibitors of extracellular heat shock protein 90 (eHSP90) have been shown to inhibit prostate cell motility. In contrast to the intracellular HSP90 (iHSP90), whose biology has been extensively studied, and whose therapeutic targeting is associated with systemic toxicity, the biology of extracellular HSP90 is not well understood. We undertook a series of investigations to better understand eHSP90 biology in PCa. Methods: Human PCa cells were subjected to cellular stress by heat shock combined with serum starvation. Resultant conditioned media was probed for matrix metalloproteinase 2 (MMP-2) by zymography and subject to scratch-wound and Boyden chamber invasion assays. Expression of HSP90 and CDC37 was measured by Western blot. Plasma from patients with PCa and healthy controls was probed for eHSP90 by ELISA. Results: We have examined expression of iHSP90 and eHSP90 after cellular stress, induced by heat shock and serum starvation, in human PC3, LNCaP, C4-2B, 1532NPTX and 1532CPTX PCa cell lines. Cell stress induces increased eHSP90 in all PCa cell lines examined. This is associated with a decrease in MMP-2 activity in conditioned media. Conditioned media from stressed cells was also shown to decrease invasion of PC3 cells, consistent with changes in MMP-2. Of high interest, the addition of protease inhibitors to conditioned media led to a complete reversal of the MMP-2 response: its activity increased with cellular stress. When measuring HSP90α in plasma from healthy controls (N = 20) and patients with PCa (N = 40, 20 localized and 20 metastatic), we demonstrated a significant (p=.001) >2 fold increase in PCa compared to control. Further, a significant increase in HSP90α was observed when low versus high T stage lesions were compared. There was no statistically significant difference in HSP90α for those with metastasis to regional lymph nodes, non-regional lymph nodes, bone, or liver. Conclusion: We demonstrate that increased eHSP90 due to cellular stress is a response observed across all PCa cell lines examined and should be considered typical. Its association with extracellular MMP-2 activity is dependent on the presence of other proteases and indicates a complex regulatory mechanism. Examination of eHSP90 in humans supports its association with aggressive disease. These factors support further investigation of underlying biology and therapeutic opportunities. Citation Format: Katelyn O'Neill, Johnny Zigmond, Raymond Bergan. The emerging role of extracellular HSP90 in prostate cancer progression to a metastatic phenotype [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 3353.

Identification and functional analysis of rare HECTD1 missense variants in human neural tube defects

Neural tube defects (NTDs) are severe malformations of the central nervous system that arise from failure of neural tube closure. HECTD1 is an E3 ubiquitin ligase required for cranial neural tube closure in mouse models. NTDs in the Hectd1 mutant mouse model are due to the failure of cranial mesenchyme morphogenesis during neural fold elevation. Our earlier research has linked increased extracellular heat shock protein 90 (eHSP90) secretion to aberrant cranial mesenchyme morphogenesis in the Hectd1 model. Furthermore, overexpression of HECTD1 suppresses stress-induced eHSP90 secretion in cell lines. In this study, we report the identification of five rare HECTD1 missense sequence variants in NTD cases. The variants were found through targeted next-generation sequencing in a Chinese cohort of 352 NTD cases and 224 ethnically matched controls. We present data showing that HECTD1 is a highly conserved gene, extremely intolerant to loss-of-function mutations and missense changes. To evaluate the functional consequences of NTD-associated missense variants, functional assays in HEK293T cells were performed to examine protein expression and the ability of HECTD1 sequence variants to suppress eHSP90 secretion. One NTD-associated variant (A1084T) had significantly reduced expression in HEK293T cells. All five NTD-associated variants (p.M392V, p.T801I, p.I906V, p.A1084T, and p.P1835L) reduced regulation of eHSP90 secretion by HECTD1, while a putative benign variant (p.P2474L) did not. These findings are the first association of HECTD1 sequence variation with NTDs in humans.

Extracellular Hsp70 Is Involved in the CXCL12/CXCR4 Pathway in Primary Human Nasal Epithelial Cells: A Preliminary Study

Background and Objectives: To date, no studies have been conducted on the interaction between extracellular heat shock protein 70 (Hsp70) and C-X-C chemokine receptor type 4 (CXCR4) in the upper airway. We aimed to evaluate the relationship between extracellular Hsp70 and CXCR4 and their role in the primary human nasal epithelium.Methods: We cultured primary human nasal epithelial (HNE) cells in an air–liquid interface. Macrogen performed single-cell quantitative polymerase chain reaction and sequencing. We conducted western blot analysis for the CXCR4 and mitogen-activated protein kinase (MAPK) pathways.Results: Extracellular Hsp70 treatment significantly increased the genetic expression and protein levels of CXCR4 in primary HNE cells. Phospho-ERK expression was increased by cotreatment with Hsp70 and CXCL12, but inhibited by pretreatment with AMD3100, a CXCR4 inhibitor. Pretreatment with an anti-Hsp70 antibody reduced phospho-ERK expression upregulation induced by cotreatment with Hsp70 and CXCL12.Conclusion: Extracellular Hsp70 participates in the activation of the CXCR4-dependent downstream signaling pathway in HNE cells. Further studies should evaluate the extracellular Hsp70-CXCL12/CXCR4 axis and the role of its components in the development of inflammatory diseases.

The effect of repeated hot water immersion on insulin sensitivity, heat shock protein 70, and inflammation in individuals with type 2 diabetes mellitus.

Repeated hot water immersion (HWI) can improve glycemic control in healthy individuals but data are limited for individuals with type 2 diabetes mellitus (T2DM). The present study investigated whether repeated HWI improves insulin sensitivity and inflammatory status and reduces plasma ([extracellular heat shock protein 70]) [eHSP70] and resting metabolic rate (RMR). Fourteen individuals with T2DM participated in this pre- versus postintervention study, with outcome measures assessed in fasted (≥12 h) and postprandial (2-h post-75 g glucose ingestion) states. HWI consisted of 1 h in 40°C water (target rectal temperature 38.5°C-39°C) repeated 8-10 times within a 14-day period. Outcome measures included insulin sensitivity, plasma [glucose], [insulin], [eHSP70], inflammatory markers, RMR, and substrate utilization. The HWI intervention increased fasted insulin sensitivity (QUICKI; P = 0.03) and lowered fasted plasma [insulin] (P = 0.04), but fasting plasma [glucose] (P = 0.83), [eHSP70] (P = 0.08), [IL-6] (P = 0.55), [IL-10] (P = 0.59), postprandial insulin sensitivity (P = 0.19), plasma [glucose] (P = 0.40), and [insulin] (P = 0.47) were not different. RMR was reduced by 6.63% (P < 0.05), although carbohydrate (P = 0.43) and fat oxidation (P = 0.99) rates were unchanged. This study shows that 8-10 HWIs within a 14-day period improved fasting insulin sensitivity and plasma [insulin] in individuals with T2DM, but not when glucose tolerance is challenged. HWI also improves metabolic efficiency (i.e., reduced RMR). Together these results could be clinically important and have implications for metabolic health outcomes and well-being in individuals with T2DM.NEW & NOTEWORTHY This is the first study to investigate repeated HWI to raise deep body temperature on insulin sensitivity, inflammation, eHSP70, and substrate utilization in individuals with T2DM. The principal novel findings were improvements in fasting insulin sensitivity and fasting plasma [insulin] but no change in fasting plasma [glucose], postprandial insulin sensitivity, plasma [insulin], or [glucose]. There was also no change in eHSP70, inflammatory status, or substrate utilization but there were reductions in RMR and oxygen consumption.

GW4869 Can Inhibit Epithelial-Mesenchymal Transition and Extracellular HSP90α in Gefitinib-Sensitive NSCLC Cells.

GW4869 is an exosomal inhibitor. It is necessary to delay the occurrence of gefitinib resistance during non-small-cell lung cancer (NSCLC) treatment. This study aimed to investigate the anti-tumor effects of GW4869 on epithelial-mesenchymal transition (EMT) and expression of extracellular heat shock protein 90α (eHSP90α) that contributes to acquired resisitance. Our study provides a new sight into the treatment of EGFR-mutated NSCLC. We performed western blotting to detect levels of EMT and eHSP90α. Wound healing and transwell assays were performed to evaluate the behavioral dynamics of EMT. A nude mouse model of HCC827 was established in vivo. GW4869 inhibited the expression of eHSP90α, EMT, invasion and migration abilities of HCC827 and PC9. GW4869 enhanced sensitivity to gefitinib in BALB/c nude mice bearing tumors of HCC827. These studies suggest that GW4869 can inhibit EMT and extracellular HSP90α, providing new strategies for enhancing gefitinib sensitivity in NSCLC.

Association of elevated extracellular HSP72 in albuminuria with systemic inflammation and disease progression in type 2 diabetic kidney disease

BackgroundSub-clinical inflammation in hyperglycemia is tied to the pathogenesis of diabetic kidney disease (DKD). Though well known for its immunostimulatory function, the significance of extracellular heat shock protein 72 (eHSP72) in DKD is not well studied. We aimed to determine the association of extracellular HSP72 with systemic inflammation and the progression of DKD, and explore its possible clinical significance in DKD. Methods160 type 2 diabetic individuals were enrolled in the study. Their anthropometric data, routine biochemical parameters, urinary renal function parameters, and blood count parameters were estimated. Plasma from patients’ blood samples were used to estimate HSP72 and interleukin 1β (IL-1β) using sandwich immunoassays. ResultsPlasma eHSP72 is elevated in DKD. Pairwise comparisons showed the drastic elevation of eHSP72 in the presence of albuminuria. A significant positive relationship was observed between plasma levels of eHSP72 and IL-1β. eHSP72 levels did not statistically differ between micro and macro-albuminuric DKD. However, it was inversely associated with estimated glomerular filtration rate, the index of disease severity, independent of age, gender, diabetes duration and absolute monocyte count. At a cutoff of 0.52 ng/ml, with sensitivity of 64.1 % and specificity of 69.2 %, plasma eHSP72 differentiated the presence of DKD in type 2 diabetics with statistical significance. ConclusionThe positive relationship of eHSP72 and IL-1β with worsening DKD likely indicates their participation in immunostimulatory pathways of renal fibrosis. eHSP72 may be closely linked to albuminuria-induced tubular injury and likely contributes to fibrotic changes in the progression of DKD. From our study, we infer the possible clinical significance of eHSP72 as a marker of sub-clinical renal damage in DKD, and the implication of IL-1β-associated mechanisms in DKD progression.

Extracellular heat shock protein 70 levels in tumour-bearing dogs and cats treated with radiation therapy and hyperthermia.

Hyperthermia is a form of a cancer treatment which is frequently applied in combination with radiotherapy (RT) to improve therapy responses and radiosensitivity. The mode of action of hyperthermia is multifactorial; the one hand by altering the amount of the blood circulation in the treated tissue, on the other hand by modulating molecular pathways involved in cell survival processes and immunogenic interactions. One of the most dominant proteins induced by hyperthermia is the major stress-inducible heat shock protein 70 (Hsp70). Hsp70 can be found in the blood either as a free-protein (free HSP70) derived from necrotic cells, or lipid-bound (liposomal Hsp70) when it is actively released in extracellular vesicles (EVs) by living cells. The aim of the study was to evaluate the levels of free and liposomal Hsp70 before and after treatment with RT alone or hyperthermia combined with radiotherapy (HTRT) in dogs and cats to evaluate therapy responses. Peripheral blood was collected from feline and canine patients before and at 2, 4, 6 and 24 h after treatment with RT or HTRT. Hsp70 enzyme-linked immunosorbent assays (ELISAs) were performed to determine the free and liposomal Hsp70 concentrations in the serum. The levels were analysed after the first fraction of radiation to study immediate effects and after all applied fractions to study cumulative effects. The levels of free and liposomal Hsp70 levels in the circulation were not affected by the first singular treatment and cumulative effects of RT in cats however, after finalizing all treatment cycles with HTRT free and liposomal Hsp70 levels significantly increased. In dogs, HTRT, but not treatment with RT alone, significantly affected liposomal Hsp70 levels during the first fraction. Free Hsp70 levels were significantly increased after RT, but not HTRT, during the first fraction in dogs. In dogs, on the other hand, RT alone resulted in a significant increase in liposomal Hsp70, but HTRT did not significantly affect the liposomal Hsp70 when cumulative effects were analysed. Free Hsp70 was significantly induced in dogs after both, RT and HTRT when cumulative effects were analysed. RT and HTRT treatments differentially affect the levels of free and liposomal Hsp70 in dogs and cats. Both forms of Hsp70 could potentially be further investigated as potential liquid biopsy markers to study responses to RT and HTRT treatment in companion animals.

Extracellular Heat Shock Protein 70 Increases the Glucocorticoid Receptor and Dual-Specificity Phosphatase 1 via Toll-like Receptor 4 and Attenuates Inflammation in Airway Epithelial Cells.

Heat shock protein 70 (HSP70) regulates the ligand binding of the glucocorticoid receptor (GR). In asthma patients, heat treatment increased both the GR expression and secretion of extracellular HSP70 (eHSP70) by bronchial epithelial cells (EC). The objective of this study was to assess the effects of eHSP70 on GR expression and the GR-dependent regulation of immune response in human bronchial ECs. Cells were treated with either eHSP70 or transfected with an expression vector for intracellular HSP70 (iHSP70). Ribonucleic acid (RNA) and protein levels were detected by reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting, and immunofluorescence. Interleukin (IL-6 and IL-8) secretion was determined by enzyme linked immunosorbent assay (ELISA). The overexpression of iHSP70 decreased, while eHSP70 increased GR expression. In addition, eHSP70 increased the expression of the GR target dual-specificity phosphatase 1 (DUSP-1). In doing so, eHSP70 reduced the tumor growth factor (TGF)-β1-dependent activation of extracellular signal-regulated kinase (Erk)-1/2 and cyclic AMP response element binding protein (CREB) and the secretion of IL-6 and IL-8. Blocking the GR or Toll-like receptor 4 (TLR4) counteracted all eHSP70-induced effects. This study demonstrates a novel anti-inflammatory effect of eHSP70 by the signaling cascade of TLR4-GR-DUSP1, which inhibits TGF-β1-activated pro-inflammatory ERK1/2-CREB signaling and cytokine secretion. The findings suggest that eHSP70 might present a novel non-steroidal therapeutic strategy to control airway inflammation in asthma.

Irisin acts through its integrin receptor in a two-step process involving extracellular Hsp90α

Exercise benefits the human body in many ways. Irisin is secreted by muscle, increased with exercise, and conveys physiological benefits, including improved cognition and resistance to neurodegeneration. Irisin acts via αV integrins; however, a mechanistic understanding of how small polypeptides like irisin can signal through integrins is poorly understood. Using mass spectrometry and cryo-EM, we demonstrate that the extracellular heat shock protein 90α (eHsp90α) is secreted by muscle with exercise and activates integrin αVβ5. This allows for high-affinity irisin binding and signaling through an Hsp90α/αV/β5 complex. By including hydrogen/deuterium exchange data, we generate and experimentally validate a 2.98Å RMSD irisin/αVβ5 complex docking model. Irisin binds very tightly to an alternative interface on αVβ5 distinct from that used by known ligands. These data elucidate a non-canonical mechanism by which a small polypeptide hormone like irisin can function through an integrin receptor.

Carbohydrate, but not fat, oxidation is reduced during moderate-intensity exercise performed in 33 vs. 18°C at matched heart rates.

Exposure to environmental heat stress increases carbohydrate oxidation and extracellular heat shock protein 70 (HSP70) concentrations during endurance exercise at matched absolute, external work rates. However, a reduction in absolute work rate typically occurs when unacclimated enduranceathletes train and/or compete in hot environments. We sought to determine the effect of environmental heat stress on carbohydrate oxidation rates and plasma HSP70 expression during exercise at matched heart rates (HR). Ten endurance-trained, male cyclists performed two experimental trials in an acute, randomised, counterbalanced cross-over design. Each trial involved a 90-min bout of cycling exercise at 95% of the HR associated with the first ventilatory threshold in either 18 (TEMP) or 33°C (HEAT), with ~ 60% relative humidity. Mean power output (17 ± 11%, P < 0.001) and whole-body energy expenditure (14 ± 8%, P < 0.001) were significantly lower in HEAT. Whole-body carbohydrate oxidation rates were significantly lower in HEAT (19 ± 11%, P = 0.002), while fat oxidation rates were not different between-trials. The heat stress-induced reduction in carbohydrate oxidation was associated with the observed reduction in power output (r = 0.64, 95% CI, 0.01, 0.91, P = 0.05) and augmented sweat rates (r = 0.85, 95% CI, 0.49, 0.96, P = 0.002). Plasma HSP70 and adrenaline concentrations were not increased with exercise in either environment. These data contribute to our understanding of how moderate environmental heat stress is likely to influence substrate oxidation and plasma HSP70 expression in an ecologically-valid model of endurance exercise.

Role of Extracellular Heat Shock Protein 90 Alpha in the Metastasis of Oral Squamous Cell Carcinoma: A Systematic Review.

Heat shock proteins (HSPs) are expressed in a variety of cancers in human beings and are correlated with differentiation, proliferation, and metastasis. Head and neck squamous cell carcinomas, like other tumors, are exposed to environmental stress, and lack of oxygen and nutrients, and in such situations, hypoxic inducible factor (HIF) initiates the expression of genes causing angiogenesis, invasion, and metastasis. Extracellular heat shock proteins 90 alpha (eHSP90α) are overexpressed in cancers leading to tumor progression and metastasis. Hence, this review will focus on the role of eHSP90α in the metastasis of oral squamous cell carcinomas (OSCC). Different online databases were scoured for relevant articles from October 2000 to October 2022. A total of 342 articles along with duplicates were excluded. The retrieved 45 articles were studied and 39 of them were found to be not eligible as they lacked intervention and their outcome measures did not match with the present review. The final qualitative evaluation included four articles that fulfilled the eligibility criterion. A definitive expression of HSP90was implicated, as seen in three studies, suggesting its probable role as a prognostic marker for OSCC, but noconclusive evidence was found. The present review suggests that eHSP90αplays a significant role in OSCC. Though a positive association was found between HSP90expression and its possible correlation with metastasis, affirmative evidence can only be derived with the conduction of many more research studies and their subsequent synthesis of results.

The Pro-Tumorigenic Role of Chemotherapy-Induced Extracellular HSP70 from Breast Cancer Cells via Intratumoral Macrophages

Tumor-associated macrophages (TAMs) contribute to tumor progression and chemoresistance; it is therefore important to clarify the altered functions of macrophages following chemotherapy. While extracellular heat shock protein (HSP) 70 is associated with therapeutic resistance, the effects of HSP70 on TAMs remain largely unknown. Here, we conducted in vitro experiments and immunohistochemistry in 116 breast carcinoma specimens to determine whether the secretion of HSP70 from breast cancer cells following chemotherapy affects macrophage function. It was revealed that the interaction of epirubicin (EPI)-exposed breast cancer cells with macrophages enhanced tumor progression, and EPI promoted the secretion of extracellular HSP70 from breast cancer cells. The expression of pro-tumorigenic macrophage marker CD163 was decreased in macrophages treated with a conditioned medium (CM) from HSP70-silenced breast cancer cells. Breast cancer cells treated with CM from HSP70-silenced breast cancer cells showed decreased expression of transforming growth factor (TGF)-β, and the pro-tumorigenic effects of macrophages were impaired when TGF-β signaling was inhibited. Immunohistochemistry demonstrated that HSP70 served as a poor prognostic factor in conjunction with macrophage infiltration. It was therefore concluded that extracellular HSP70 levels increased following chemotherapy and enhanced the pro-tumorigenic effects of TAMs, either directly or indirectly, by regulating TGF-β expression in breast cancer cells.

A Modified Differential Centrifugation Protocol for Isolation and Quantitation of Extracellular Heat Shock Protein 90 (eHsp90).

Studies of the past 15years have revealed a critical role for extracellular heat shock protein 90alpha (eHsp90α) in the development of several human disorders, including wound healing, cachexia (muscle wasting), inflammatory diseases, and cancers. The two established functions of highly purified eHsp90α protein are to promote cell survival and to stimulate cell migration. However, the mechanism of secretion and the method of isolation of eHsp90α remained to be standardized. Among the half a dozen reported methodologies, differential centrifugation is considered the "gold standard" largely for its quantitative recovery of eHsp90α from a conditioned medium of cultured cells. Herein, we describe a revised protocol that isolates three fractions of extracellular vesicles with distinct ranges of diameters and the leftover vesicle-free supernatant for biochemical analyses, especially eHsp90α, from tumor cell-conditioned media. Quantitation of the relative amount of eHsp90α can be carried out with known amounts of recombinant Hsp90α protein on the same SDS-PAGE. We believe that this modified methodology will prove to be a useful tool for studying eHsp90α in cultured cells and beyond.

Extracellular Heat Shock Protein 27 Is Released by Plasma-Treated Ovarian Cancer Cells and Affects THP-1 Monocyte Activity

Heat shock protein 27 (Hsp27) is a cytoprotective molecule and is inducible via oxidative stress. Anti-cancer therapies, such as the recently investigated gas plasma, subject tumor cells to a plethora of reactive oxygen species (ROS). In ovarian tumor microenvironments (TME), immune cells such as monocytes and macrophages can be found in large numbers and are often associated with cancer progression. Therefore, we quantified extracellular Hsp27 of OVCAR-3 and SK-OV-3 cells after gas plasma exposure in vitro. We found Hsp27 to be significantly increased. Following this, we investigated the effects of Hsp27 on THP-1 monocytes. Live cell imaging of Hsp27-treated THP-1 cells showed decelerated cell numbers and a reduction in cell cluster sizes. In addition, reduced metabolic activity and proliferation were identified using flow cytometry. Mitochondrial ROS production decreased. Using multicolor flow cytometry, the expression profile of eight out of twelve investigated cell surface markers was significantly modulated in Hsp27-treated THP-1 cells. A significantly decreased release of IL18 accommodated this. Taken together, our results suggest an immunomodulatory effect of Hsp27 on THP-1 monocytes. These data call for further investigations on Hsp27’s impact on the interplay of ovarian cancer cells and monocytes/macrophages under oxidative stress conditions.

Extracellular HSP90α promotes cellular senescence by modulating TGF-β signaling in pulmonary fibrosis.

Recent findings suggest that extracellular heat shock protein 90α (eHSP90α) promotes pulmonary fibrosis, but the underlying mechanisms are not well understood. Aging, especially cellular senescence, is a critical risk factor for idiopathic pulmonary fibrosis (IPF). Here, we aim to investigate the role of eHSP90α on cellular senescence in IPF. Our results found that eHSP90α was upregulated in bleomycin (BLM)-induced mice, which correlated with the expression of senescence markers. This increase in eHSP90α mediated fibroblast senescence and facilitated mitochondrial dysfunction. eHSP90α activated TGF-β signaling through the phosphorylation of the SMAD complex. The SMAD complex binding to p53 and p21 promoters triggered their transcription. In vivo, the blockade of eHSP90α with 1G6-D7, a specific eHSP90α antibody, in old mice attenuated the BLM-induced lung fibrosis. Our findings elucidate a crucial mechanism underlying eHSP90α-induced cellular senescence, providing a framework for aging-related fibrosis interventions.

Extracellular Hsp90α Supports the ePKM2-GRP78-AKT Axis to Promote Tumor Metastasis.

Tumor-secreted proteins can provide numerous molecular targets for cancer diagnosis and treatment. Of note, pyruvate kinase M2 (PKM2) is secreted by tumor cells to promote malignant progression, while its regulatory mechanism or the interacting network remains uncovered. In the present study, we identified extracellular heat shock protein 90 alpha (eHsp90α) as one potential interacting protein of ePKM2 by mass spectrometry (MS), which was further verified by pull-down and co-immunoprecipitation analysis. Later, we found that eHsp90α enhanced the effect of ePKM2 on migration and invasion of lung cancer cells. Blocking of Hsp90α activity, on the other hand, attenuated tumor migration or invasion induced by ePKM2. Eventually, the in vivo role of Hsp90α in regulating ePKM2 activity was validated by the mouse xenograft tumor model. Mechanistically, we found that eHsp90α binds to and stabilizes ePKM2 to protect it from degradation in the extracellular environment. Besides, eHsp90α promoted the interaction of ePKM2 with cell surface receptor GRP78, which leads to the activation of the ePKM2/GRP78/AKT axis. Collectively, we unraveled the novel molecular mechanism of eHsp90α in regulating ePKM2 activity during tumor progression, which is beneficial for the development of new treatments against lung cancer.

Secreted HSP90α-LRP1 Signaling Promotes Tumor Metastasis and Chemoresistance in Pancreatic Cancer.

The extracellular heat shock protein 90α (eHSP90α) has been reported to promote cancer cell motility. However, whether pancreatic cancer (PC) cells expressed membrane-bound or secreted HSP90α, as well as its underlying mechanism for PC progression, were still unclear. Our study demonstrated that the amounts of secreted HSP90α proteins were discrepant in multiple PC cells. In addition, highly invasive Capan-2 cells have a higher level of secreted HSP90α compared with those of less invasive PL45 cells. The conditioned medium of Capan-2 cells or recombinant HSP90α treatment stimulated the migration and invasion of PC cells, which could be prevented with a neutralizing anti-HSP90α antibody. Furthermore, secreted HSP90α promoted elements of epithelial–mesenchymal transition in PL45 cells, including increases in vimentin and Snail expressions, decreases in E-cadherin expression, and changes in cell shape towards a mesenchymal phenotype, but these phenomena were reversed by the anti-HSP90α antibody in Capan-2 cells. In addition, high levels of low-density lipoprotein receptor-related protein 1 (LRP1) were associated with worsened patient survival in pancreatic adenocarcinoma. We demonstrated LRP1 as a receptor of eHSP90α for its stimulatory role in metastasis, by activating the AKT pathway. In addition, silencing LRP1 enhanced the chemosensitivity to gemcitabine and doxorubicin in Capan-2 cells. Therefore, our study indicated that blocking secreted HSP90α underlies an aspect of metastasis and chemoresistance in PC.

Transcriptomic and Proteomic Analysis of CRISPR/Cas9-Mediated ARC-Knockout HEK293 Cells.

Arc/Arg3.1 (activity-regulated cytoskeletal-associated protein (ARC)) is a critical regulator of long-term synaptic plasticity and is involved in the pathophysiology of schizophrenia. The functions and mechanisms of human ARC action are poorly understood and worthy of further investigation. To investigate the function of the ARC gene in vitro, we generated an ARC-knockout (KO) HEK293 cell line via CRISPR/Cas9-mediated gene editing and conducted RNA sequencing and label-free LC-MS/MS analysis to identify the differentially expressed genes and proteins in isogenic ARC-KO HEK293 cells. Furthermore, we used bioluminescence resonance energy transfer (BRET) assays to detect interactions between the ARC protein and differentially expressed proteins. Genetic deletion of ARC disturbed multiple genes involved in the extracellular matrix and synaptic membrane. Seven proteins (HSPA1A, ENO1, VCP, HMGCS1, ALDH1B1, FSCN1, and HINT2) were found to be differentially expressed between ARC-KO cells and ARC wild-type cells. BRET assay results showed that ARC interacted with PSD95 and HSPA1A. Overall, we found that ARC regulates the differential expression of genes involved in the extracellular matrix, synaptic membrane, and heat shock protein family. The transcriptomic and proteomic profiles of ARC-KO HEK293 cells presented here provide new evidence for the mechanisms underlying the effects of ARC and molecular pathways involved in schizophrenia pathophysiology.