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. 2021 Mar 25;18(3):693-704.
doi: 10.20892/j.issn.2095-3941.2020.0262. Online ahead of print.

Heat shock protein 90 promotes RNA helicase DDX5 accumulation and exacerbates hepatocellular carcinoma by inhibiting autophagy

Ting Zhang #  1   2 , Xinrui Yang #  1 , Wanping Xu  3 , Jing Wang  4 , Dawei Wu  1 , Zhixian Hong  1 , Shengxian Yuan  5 , Zhen Zeng  1 , Xiaodong Jia  1 , Shanshan Lu  1 , Rifaat Safadi  6 , Sen Han  2 , Zhihong Yang  2 , Leonard M Neckers  4 , Suthat Liangpunsakul  2   7 , Weiping Zhou  5 , Yinying Lu  1   8
Affiliations

Heat shock protein 90 promotes RNA helicase DDX5 accumulation and exacerbates hepatocellular carcinoma by inhibiting autophagy

Ting Zhang et al. Cancer Biol Med. .

Abstract

Objective: Hepatocellular carcinoma (HCC), the main type of liver cancer, has a high morbidity and mortality, and a poor prognosis. RNA helicase DDX5, which acts as a transcriptional co-regulator, is overexpressed in most malignant tumors and promotes cancer cell growth. Heat shock protein 90 (HSP90) is an important molecular chaperone in the conformational maturation and stabilization of numerous proteins involved in cell growth or survival.

Methods: DDX5 mRNA and protein expression in surgically resected HCC tissues from 24 Asian patients were detected by quantitative real-time PCR and Western blot, respectively. The interaction of DDX5-HSP90 was determined by molecular docking, immunoprecipitation, and laser scanning confocal microscopy. The autophagy signal was detected by Western blot. The cell functions and signaling pathways of DDX5 were determined in 2 HCC cell lines. Two different murine HCC xenograft models were used to determine the function of DDX5 and the therapeutic effect of an HSP90 inhibitor.

Results: HSP90 interacted directly with DDX5 and inhibited DDX5 protein degradation in the AMPK/ULK1-regulated autophagy pathway. The subsequent accumulation of DDX5 protein induced the malignant phenotype of HCC by activating the β-catenin signaling pathway. The silencing of DDX5 or treatment with HSP90 inhibitor both blocked in vivo tumor growth in a murine HCC xenograft model. High levels of HSP90 and DDX5 protein were associated with poor prognoses.

Conclusions: HSP90 interacted with DDX5 protein and subsequently protected DDX5 protein from AMPK/ULK1-regulated autophagic degradation. DDX5 and HSP90 are therefore potential therapeutic targets for HCC.

Keywords: Hepatocellular carcinoma; RNA helicase DDX5; autophagy; heat shock protein 90; β-catenin pathway.

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Conflict of interest statement

No potential conflicts of interest are disclosed.

Figures

Figure 1
Figure 1
DDX5 mRNA and protein expression in patients with hepatocellular carcinoma (HCC) and its prognostic significance. (A) Relative expression of DDX5 mRNA in HCC using The Cancer Genome Atlas database from 371 HCC tissues compared to 50 non-neoplastic liver tissues; P < 0.001. (B) DDX5 and HSP90 protein expressions in HCC tissues (T) and surrounding non-neoplastic area (NT) detected by Western blot; *P < 0.05; ***P < 0.001. (C) Immunohistochemistry of DDX5 from HCC and non-neoplastic tissues (Scale bar: 100 μm). (D) The mRNA level of DDX5 in HCC surgical specimens detected by qRT-PCR (mean ± SEM, n = 24, P = 0.008). (E) The incidence of HCC recurrence after resection stratified by baseline DDX5 expressions in tumor tissues (cut-off value, T/NT= 1, n = 24, P = 0.04).
Figure 2
Figure 2
DDX5 interacts directly with HSP90. (A) Molecular docking between DDX5 and HSP90. The blue area represents HSP90, and the red area represents DDX5. The green and orange areas (arrows) represent the interaction between HSP90 and DDX5. (B and C) Co-immunoprecipitation between HSP90 with overexpressed DDX5-FLAG and endogenous DDX5. Lane IgG was a negative control; Lane IP was conducted using antibodies for FLAG (upper panel) or DDX5 (lower panel) antibodies; Lane input was the cell lysate (as a positive control). (D) Subcellular co-localization of DDX5 and HSP90 using confocal microscopy (Scale bar: 10 μm). HepG2 cells were stained with DAPI (blue, nuclear staining), DDX5 antibody (red), and HSP90 antibody (green). (E) A FLAG pull-down assay of truncated DDX5 (1-122 amino acids)-FLAG fusion fragment. Lane 2 is a double loading of lane 1.
Figure 3
Figure 3
HSP90 inhibits the autophagic degradation of DDX5 protein. (A) The mRNA level of DDX5 after HSP90 knockdown with shRNA (Hsp90-K.D.), tested by qRT-PCR, using GAPDH as a reference gene. (B) The protein level of DDX5 after HSP90 knockdown (Hsp90-K.D.) (mean ± SD, *P = 0.0238). (C) The mRNA level of DDX5 after STA9090 treatment, tested by qRT-PCR, using GAPDH as a reference gene. (D) The protein level of DDX5 after STA9090 treatment (mean ± SD, *P = 0.0294). (E) The inhibitory effect of DDX5 expression in the presence of STA9090, an inhibitor of HSP90, was ameliorated in HepG2 cells treated with an inhibitor for autophagy, MRT68921, but not by MG132, an inhibitor of proteasomes. (F) Confocal microscopy of intracellular localization analysis of DDX5 and autophagosomes after treatment with STA9090, chloroquine, and AICAR (Scale bar: 10 μm). Chloroquine: inhibitor of lysosome; AICAR: agonist of autophagy. Arrow: DDX5 combined with autophagosomes.
Figure 4
Figure 4
HSP90 inhibits DDX5 degradation and increases its expression by inhibition of the AMPK/ULK1-regulated autophagic pathway. (A) Western blot analysis of the protein expressions of AMPK, p-AMPK, ULK1, p-ULK1, Beclin1, LC3II, ATG5, and DDX5 in HepG2 cells treated with AICAR (positive control) STA9090 (a HSP90 inhibitor), and HSP90 knockdown (Hsp90-K.D). (B–E) Inhibition of DDX5 decreases cellular viability, migration, and invasion in the hepatoma cell line. (B) The cell viability of both HepG2 and Huh7 cell lines determined by the CCK8 assay. Circle, control group; square, STA9090 treatment group; upper triangle, DDX5 knockdown group (DDX5-K.D.); down triangle, the group treated with both DDX5-K.D. and STA9090. (C) Lack of DDX5 (DDX5-K.D. or STA9090 treatment) inhibited cellular proliferation in both HepG2 and Huh7 cells. (D) Cellular migration (D) and invasion (E) was inhibited in both Huh7 and HepG2 cells in DDX5 KD or STA9090 treatment (Scale bars, 200 μm). Data are from a representative experiment that was repeated 3 times with similar results (mean ± SD, *P < 0.05; **P < 0.01; ***P < 0.001).
Figure 5
Figure 5
DDX5 promotes the malignant phenotype of hepatocellular carcinoma by activating the β-catenin signaling pathway. (A) Western blot analysis of DDX5 protein expressions in HepG2 and Huh7 cells, (total and nuclear) β-catenin, c-Myc, and cyclin D1 in groups of control, DDX5-K.D. and STA9090 treatments, respectively. GAPDH was used as an internal reference of total proteins, and Histone H3 was used as an internal reference of nuclear proteins; *P < 0.05; **P < 0.01; ***P < 0.001. (B) Immunofluorescence analysis of β-catenin expression in DDX5-K.D. (Scale bar: 10 μm). (C) DDX5-K.D. decreased the Top/Fop Flash activity (mean ± SD, P = 0.006).
Figure 6
Figure 6
Knockdown of DDX5 blocks in vivo tumor growth in a murine hepatocellular carcinoma xenograft model. (A) DDX5-K.D. inhibited the tumor growth of the Huh7 mice xenograft model (left, photograph of tumors, scale bar: 10 mm; right, tumor volume, mean ± SD, ***P < 0.001). (B) STA9090 inhibited tumor growth in the Huh7 mice xenograft model (left, photograph of tumors, scale bar: 10 mm; right, tumor volume, mean ± SD, ***P < 0.001). (C) Immunohistochemical analysis of DDX5 in different groups of xenograft models (Scale bar: 50 μm). (D) Immunoblot analysis of DDX5 expression and the β-catenin signal pathway, and c-Myc and cyclin D1 expressions in STA9090-treated Huh7 cells and the DDX5-K.D. Huh7 xenograft mice models. Data are from a representative experiment that was repeated 3 times with similar results (mean ± SD, *P < 0.05; **P < 0.01; ***P < 0.001). (E) The schematic pathway summarizes the role of HSP90-DDX5 in promoting HCC. In HSP90++ HCC, the HSP90 inhibits DDX5 degradation and increases its expression through inhibition of autophagic pathway. DDX5 promotes the malignant phenotype of HCC by activating the β-catenin pathway.
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