HSP90 N-terminal inhibition promotes mitochondria-derived vesicles related metastasis by reducing TFEB transcription via decreased HSP90AA1-HCFC1 interaction in liver cancer.

Cancer cells compensate with increasing mitochondria-derived vesicles (MDVs) to maintain mitochondrial homeostasis, when canonical MAP1LC3B/LC3B (microtubule associated protein 1 light chain 3 beta)-mediated mitophagy is lacking. MDVs promote the transport of mitochondrial components into extracellular vesicles (EVs) and induce tumor metastasis. Although HSP90 (heat shock protein 90) chaperones hundreds of client proteins and its inhibitors suppress tumors, HSP90 inhibitors-related chemotherapy is associated with unexpected metastasis. Herein, we find that HSP90 inhibitor causes mitochondrial damage but stimulates the low LC3-induced MDVs and the release of MDVs-derived EVs. However, why LC3 decreases and what is the transcriptional regulatory mechanism of MDVs formation under HSP90 inhibition remain unknown. Because TFEB (transcription factor EB) is the most important mitophagy transcription factor, and the HSP90 client HCFC1 (host cell factor C1) regulates TFEB transcription, there should be a hidden connection between TFEB, HCFC1 and HSP90 in MDVs formation. Our results support the idea that HSP90 N-terminal inhibition reduces TFEB transcription via decreased HSP90AA1-HCFC1 interaction, which prevents HCFC1 from binding to the TFEB proximal promoter region. Decreased TFEB transcription and consequently reduced LC3, ultimately promoted MDVs formation. Blocking MDVs formation with the microtubule inhibitor nocodazole (NOC) activates the HCFC1-TFEB-LC3 axis, weakens HSP90 inhibitors-induced MDVs and the release of MDVs-derived EVs, inhibits the growth of tumor cell spheres and primary liver tumors, and reduces the extravasation of cancer cells to secondary metastatic sites. Taken together, these data suggest that combination therapy should be used to reduce the metastatic risk of low TFEB-triggered-MDVs formation caused by HSP90 inhibitors.

HSP90 C-terminal domain inhibition promotes VDAC1 oligomerization via decreasing K274 mono-ubiquitination in Hepatocellular Carcinoma.

Voltage-dependent anion-selective channel protein 1 (VDAC1) is the most abundant protein in the mitochondrial outer membrane and plays a crucial role in the control of hepatocellular carcinoma (HCC) progress. Our previous research found that cytosolic molecular chaperone heat shock protein 90 (Hsp90) interacted with VDAC1, but the effect of the C-terminal and N-terminal domains of Hsp90 on the formation of VDAC1 oligomers is unclear. In this study, we focused on the effect of the C-terminal domain of Hsp90 on VDAC1 oligomerization, ubiquitination, and VDAC1 channel activity. We found that Hsp90 C-terminal domain inhibitor Novobiocin promoted VDAC1 oligomerization, release of cytochrome c, and activated mitochondrial apoptosis pathway. Atomic coarse particle modeling simulation revealed C-terminal domain of Hsp90α stabilized VDAC1 monomers. The purified VDAC1 was reconstituted into a planar lipid bilayer, and electrophysiology experiments of patch clamp showed that the Hsp90 C-terminal inhibitor Novobiocin increased VDAC1 channel conductance via promoting VDAC1 oligomerization. The mitochondrial ubiquitination proteomics results showed that VDAC1 K274 mono-ubiquitination was significantly decreased upon Novobiocin treatment. Site-directed mutation of VDAC1 (K274R) weakened Hsp90α-VDAC1 interaction and increased VDAC1 oligomerization. Taken together, our results reveal that Hsp90 C-terminal domain inhibition promotes VDAC1 oligomerization and VDAC1 channel conductance by decreasing VDAC1 K274 mono- ubiquitination, which provides a new perspective for mitochondria-targeted therapy of HCC.

Mechanical Behaviors of Microwave-Assisted Pyrolysis Recycled Carbon Fiber-Reinforced Concrete with Early-Strength Cement.

This study aimed to investigate the mechanical performance of early-strength carbon fiber-reinforced concrete (ECFRC) by incorporating original carbon fiber (OCF), recycled carbon fiber (RCF), and sizing-removed carbon fiber (SCF). Compressive, flexural, and splitting tensile strength were tested under three fiber-to-cement weight ratios (5‱, 10‱, and 15‱). The RCF was produced from waste bicycle parts made of carbon fiber-reinforced polymer (CFRP) through microwave-assisted pyrolysis (MAP). The sizing-removed fiber was obtained through a heat-treatment method applied to the OCF. The results of scanning electron microscopy (SEM) analysis with energy dispersive X-ray spectrometry (EDS) indicated the successful removal of sizing and impurities from the surface of the RCF and SCF. The mechanical test results showed that ECFRC with a 10‱ fiber-to-cement weight ratio of carbon fiber had the greatest improvement in its mechanical strengths. Moreover, the ECFRC with 10‱ RCF exhibited higher compressive, flexural, and splitting tensile strength than that of benchmark specimen by 14.2%, 56.5%, and 22.5%, respectively. The ECFRC specimens with a 10‱ fiber-to-cement weight ratio were used to analyze their impact resistance under various impact energies in the impact test. At 50 joules of impact energy, the impact number of the ECFRC with SCF was over 23 times that of the benchmark specimen (early-strength concrete without fiber) and was also greater than that of ECFRC with OCF and RCF.

Hsp90 Inhibitor STA9090 induced VPS35 related extracellular vesicle release and metastasis in hepatocellular carcinoma.

Heat shock protein 90 (Hsp90) has been an important therapeutic target for cancer therapy for decades. Unexpectedly, the monotherapy of N-terminal Hsp90 inhibitor STA9090 related clinical trials halted in phase III, and metastases were reported in animal models with the treatment of N-terminal Hsp90 inhibitors. Vacuolar protein sorting-associated protein 35 (VPS35) plays a vital role in endosome-derived EV (extracellular vesicle) traffic in neurodegeneration diseases, but no vps35 related EV were reported in tumors till now. Since tumor derived EVs contributes to metastasis and VPS35 is recently found to be involved in the invasion and metastasis of hepatocellular carcinoma (HCC), whether N-terminal Hsp90 inhibitor STA9090 induced EVs generation and the role of VPS35 in it were explored in this study. We found that N-terminal Hsp90 inhibitor STA9090 upregulated Bclaf1 and VPS35 levels, increased the secretion of EVs, and STA9090-induced-EVs promoted the invasion of HepG2 cells. As the clinical data suggested that the increased Bclaf1 and VPS35 levels correlated with increased metastasis and poorer prognosis in HCC, we focused on the Bclaf1-VPS35-EVs axis to further explore the mechanism of VPS35-related metastasis. The results demonstrated that Bclaf1 facilitated the transcription of VPS35 via bZIP domain, and knockdown of Bclaf1 or VPS35 alleviated pro-metastatic capability of STA9090-induced-EVs. All the results revealed the role of Bclaf1-VPS35-EVs axis on metastasis of HCC, and VPS35 knockdown decreased Hsp90 Inhibitor STA9090 induced extracellular vesicle release and metastasis, which provided a new combination therapeutic strategy to inhibit the metastasis of HCC caused by N-terminal Hsp90 inhibitor induced extracellular vesicles.

Extracellular Hsp90α, which participates in vascular inflammation, is a novel serum predictor of atherosclerosis in type 2 diabetes.

INTRODUCTION: Atherosclerosis is the main pathological change in diabetic angiopathy, and vascular inflammation plays an important role in early atherosclerosis. Extracellular heat shock protein 90 (eHsp90) is secreted into the serum and is involved in various physiological and pathophysiological processes. However, the specific mechanism of eHsp90 in early atherosclerosis remains unclear. This study explored the relationship between Hsp90 and diabetic lower extremity arterial disease and investigated the expression of eHsp90 in vascular endothelial cells under environmental stimulation and the function and mechanism of eHsp90α involved in diabetic atherosclerosis. RESEARCH DESIGN AND METHODS: One hundred and three selected patients were divided into three groups: the diabetes mellitus group (n=27), the diabetic lower extremity arterial disease group (n=46), and the diabetic critical limb ischemia group (n=30). The relationships among serum Hsp90, oxidative stress indexes, and patient outcomes and the correlations among the indexes were analyzed. H&E staining and immunohistochemistry were used to observe the vasculature of amputated feet from patients with diabetic foot. An oxidative stress endothelial injury model was established under high glucose in vitro to explore the role of eHsp90 release in atherosclerosis progression. RESULTS: The level of serum Hsp90 was upregulated with aggravation of diabetic vascular disease. Hsp90α was correlated with malondialdehyde to some extent and was an independent risk factor in the progression of diabetic vascular disease, with predictive ability. The expression area of Hsp90α was consistent with the area of inflammatory infiltration in the vessel lumen. Vascular endothelial cells were found to increase eHsp90α secretion under stress. Then inhibition of eHsp90α can reduce the degree of cellular inflammation and damage. Endothelial cell-conditioned medium and recombinant human Hsp90α increased monocyte migration via the low-denisity lipoprotein receptor-related protein 1 (LRP1) receptor to promote disease progression. CONCLUSIONS: eHsp90α plays a critical role in the early inflammatory injury stage of atherosclerosis. TRIAL REGISTRATION NUMBER: NCT04787770.

Hsp90 Inhibitor STA9090 Sensitizes Hepatocellular Carcinoma to Hyperthermia-Induced DNA Damage by Suppressing DNA-PKcs Protein Stability and mRNA Transcription.

As a conserved molecular chaperone, heat shock protein 90 (Hsp90) maintains the stability and homeostasis of oncoproteins and helps cancer cells survive. DNA-dependent protein kinase catalytic subunit (DNA-PKcs) plays a pivotal role in the non-homologous end joining pathway for DNA double-strand breaks (DSB) repair. Tumor cells contain higher levels of DNA-PKcs to survive by the hostile tumor microenvironment and various antitumor therapies. Here, we showed that increased levels of Hsp90α, Hsp90ß, and DNA-PKcs correlated with a poor overall survival in hepatocellular carcinoma (HCC). We revealed that Hsp90 N-terminal domain and C-terminal domain have different effects on DNA-PKcs protein and mRNA levels. The stability of DNA-PKcs depended on Hsp90α N-terminal nucleotide binding domain. Transcription factor SP1 regulates the transcription of PRKDC (gene name of DNA-PKcs) and is a client protein of Hsp90. Inhibition of Hsp90 N-terminal by STA9090 decreased the location of Hsp90α in nucleus, Hsp90α-SP1 interaction, SP1 level, and the binding of Hsp90α/SP1 at the proximal promoter region of PRKDC Because hyperthermia induces DSBs with increases level of DNA-PKcs, combined STA9090 treatment with hyperthermia effectively delayed the tumor growth and significantly decreased DNA-PKcs levels in xenografts model. Consistently, inhibition of Hsp90 increased the number of heat shock-induced γ-H2AX foci and delayed the repair of DSBs. Altogether, our results suggest that Hsp90 inhibitor STA9090 decreases DNA-PKcs protein stability and PRKDC mRNA level, which provide a theoretical basis for the promising combination therapy of hyperthermia and Hsp90 inhibitor in HCC.

E4F1 silencing inhibits the cell growth through cell-cycle arrest in malignant transformed cells induced by hydroquinone.

Hydroquinone (HQ), one of the most significant metabolic activation products of benzene in an organism, can cause hematological toxicity, such as acute myeloid leukemia. It is a clear carcinogen that can cause changes in the disorder of cell cycle and cell growth. However, its molecular mechanisms remain unclear. E4 transcription factor 1 (E4F1), an important transcription factor, participating in the regulation of cell cycle may be related to the occurrence of tumor. Here, we examined the HQ-induced malignant transformed TK6 cells (TK6-HT) to illustrate the role of E4F1 in carcinogenesis. The present study showed that both the expressions of E4F1 messenger RNA and protein increased obviously in TK6-HT, preliminarily indicating that E4F1 is associated with HQ-induced carcinogenesis. To further explore the role of E4F1, we established E4F1 silencing TK6-HT (pLVX-shE4F1) and its control cells (pLVX-shNC) using lentiviral short hairpin RNA (shRNA) interference expression plasmid vector pLVX-shRNA. Flow cytometry and cell counting kit-8 assay were used to determine the effects of E4F1 silencing on cell cycle and cell growth, respectively. E4F1 silencing inhibited cell growth in TK6-HT. The results from flow cytometry indicated that the inhibitory effect on cell growth may be the results of the E4F1 silencing-induced accumulation in G2/M compared with TK6-HT-shNC. Meanwhile, levels of DNA damage (γ-H2AX), proteins of Rb and phosphorylated Rb, and reactive oxygen species were increased in TK6-HT-shRNA2 cells, which is the critical reason of cell-cycle arrest. In conclusion, E4F1 silencing inhibits the cell growth through cell-cycle arrest in malignant transformed cells induced by HQ.

Poly(ADP-ribose) polymerase-1 promotes expression of miR-155 by the up-regulation of methyl-CpG binding domain protein 2 in TK6 cells exposed to hydroquinone.

Hydroquinone (HQ), one of the major metabolites of benzene, can induce aberrant gene expression. MiR-155, a tumor activator, participates in various biological processes, including DNA damage response. However, the molecular mechanism of aberrant miR-155 expression is still not completely elucidated. Here, we investigated the mechanism of abnormal expression of miR-155 induced by poly(ADP-ribose)polymerase-1 (PARP-1) expression in HQ-treated TK6 lymphoblastoid cells. We examined the expression of genes related to abnormal expression of miR-155 to explore the reason for this phenomenon. The results of the present study showed that miR-155 was significantly increased and reactive oxygen species (ROS) were decreased in cells treated with HQ for 72 h compared with PBS-treated cells. Meanwhile, E4F1, PARP-1 and PARP-1 related co-regulators (NF-κB, HDAC1, and HDAC2), acetylated histone H3 (H3Ac) were increased in a concentration-dependent manner. Experiments for treatment with 5-AzaC (DNMTs inhibitor), TSA (HDACs inhibitor), DOX (to activate PARP-1) or MG132 (proteasome inhibitor) revealed that the MBDs and PARP-1 was positively associated with miR-155 expression. Moreover, in cells treated with HQ in conjunction with PARP-1 knockdown, expression of miR-155, H3Ac and MBD2 protein were decreased, compared with negative control. In conclusion, PARP-1 activates expression of miR-155 via acetylation by regulating MBD2 in TK6 cells exposed to HQ.