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.

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.

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.