DCAF1 interacts with PARD3 to promote hepatocellular carcinoma progression and metastasis by activating the Akt signaling pathway

Abstract

Background: Hepatocellular carcinoma (HCC) is a fatal malignancy with poor prognosis due to lack of effective clinical interference. DCAF1 plays a vital role in regulating cell growth and proliferation, and is involved in the progression of various malignancies. However, the function of DCAF1 in HCC development and the underlying mechanism are still unknown. This study aimed to explore the effect of DCAF1 in HCC and the corresponding molecular mechanism.

Methods: Quantitative real-time PCR, Western blot and immunostaining were used to determine DCAF1 expression in tumor tissues and cell lines. Subsequently, in vitro and in vivo experiments were conducted to explore the function of DCAF1 in tumor growth and metastasis in HCC. Coimmunoprecipitation, mass spectrometry and RNA sequencing were performed to identify the underlying molecular mechanisms.

Results: In this study, we found that DCAF1 was observably upregulated and associated with poor prognosis in HCC. Knockdown of DCAF1 inhibited tumor proliferation and metastasis and promoted tumor apoptosis, whereas overexpressing DCAF1 yielded opposite effects. Mechanistically, DCAF1 could activate the Akt signaling pathway by binding to PARD3 and enhancing its expression. We also found that the combined application of DCAF1 knockdown and Akt inhibitor could significantly suppress subcutaneous xenograft tumor growth.

Conclusions: Our study illustrates that DCAF1 plays a crucial role in HCC development and the DCAF1/PARD3/Akt axis presents a potentially effective therapeutic strategy for HCC.

Keywords: Akt; DCAF1; Hepatocellular carcinoma; PARD3.

Fig. 1

DCAF1 is upregulated in HCC tissues and correlated with poor prognosis. A Related mRNA expression of DCAF1 in HCC and adjacent peritumor tissues tested by qRT‒PCR. B Related mRNA expression of DCAF1 in the public dataset GSE63898. C Related protein expression of DCAF1 in tumor (T), adjacent peritumor (P) and normal (N) tissues tested by Western blot. D-E DCAF1 expression in HCC tissues and adjacent peritumor tissues was tested by IF D and IHC E assays (Bar = 50μm(D), Bar = 100μm(E)). F Kaplan–Meier analysis of the overall survival of patients from the DCAF1 high and low expression groups in the TCGA-LIHC dataset. * p < 0.05, *** p < 0.001

Fig. 2

DCAF1 promotes HCC proliferation in vitro and in vivo. A Expression of DCAF1 in HCC cell lines and HepRG cells detected by Western blot and qRT‒PCR. B The efficacy of DCAF1 knockdown in Hep3B and SMMC-7721 cells verified by Western blot. C The colony formation results of Hep3B and SMMC-7721 cells after knockdown of DCAF1. D EdU assays results of Hep3B and SMMC-7721 cells after DCAF1 knockdown (Bar = 100μm). E CCK8 assays of Hep3B and SMMC-7721 cells after knockdown of DCAF1. F Cell cycle analysis of Hep3B and SMMC-7721 cells was conducted by flow cytometry. G The cell apoptosis ratio of Hep3B and SMMC-7721 cells was detected by flow cytometry using the Annexin V-FITC/PI staining kit. H Hep3B cells with DCAF1 knockdown were transplanted on nude mice, and tumor volumes and tumor weights were recorded. I SMMC-7721 cells with DCAF1 knockdown were transplanted on nude mice, and tumor volumes and tumor weights were recorded. Data are shown as the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Fig. 3

DCAF1 regulates cell migration and invasion in HCC cell lines and activates EMT. A Representative image of migrated and invaded Hep3B and SMMC-7721 cells after DCAF1 knockdown (Bar = 100μm). B Wound healing assays were performed to assess the effect of DCAF1 knockdown on cell motility in HCC cells (Bar = 500μm). C The protein expression levels of EMT markers in Hep3B and SMMC-7721 cells after knockdown of DCAF1 were detected by Western blot. D The mRNA expression levels of EMT markers in Hep3B and SMMC-7721 cells after knockdown of DCAF1 were detected by qRT‒PCR. Data are shown as the mean ± SD. E The expression of EMT marker proteins in xenografts was assessed by Western blot. ** p < 0.01, *** p < 0.001, **** p < 0.0001

Fig. 4

DCAF1 associates with and upregulates PARD3 to promote HCC growth. A The top 5 proteins associated with DCAF1 detected by LC–MS/MS. B, C Association of DCAF1 and PARD3 verified by coIP. D DCAF1 C-terminus containing residues 1339–1507 was found to be responsible for the binding of DCAF1 to PARD3. E Western blot results suggested that PARD3 was positively regulated by DCAF1 at the protein level. F The expression level of PARD3 in HepG2 cells was verified by Western blot. G-J PARD3 partly repressed the promoting effects on HCC cells proliferation and migration caused by DCAF1 overexpression. colony formation assays G and CCK8 assays H indicated that PARD3 could partly inhibit the promoting effects of DCAF1 overexpression on cell proliferation. Transwell assays I and wound healing assays J demonstrated that the knockdown of PARD3 partly reversed the promoting effects on cell migration and invasion caused by the overexpression of DCAF1 (Bar = 100μm (I), Bar = 500μm (J)). Data are shown as the mean ± SD. ** p < 0.01, *** p < 0.001, **** p < 0.0001

Fig. 5

DCAF1 activates the Akt signaling pathway through regulating PARD3. A The volcano plots of differentially expressed genes in Hep3B and SMMC-7721 cells transfected with PARD3-shRNA and control vector. Significantly upregulated genes are denoted in red, significantly downregulated genes in blue, and non-significant changes in gray. B The Venn grams of downregulated genes in Hep3B and SMMC-7721 cells. C KEGG pathway analysis of downregulated genes in Hep3B and SMMC-7721 cells. D The protein levels of Akt and p-Akt in HepG2 cells transfected with PARD3-shRNA or control vector were tested by Western blot. E The protein levels of Akt and p-Akt in Hep3B cells with PARD3 overexpression or pCDH vector were tested by Western blot. F The protein levels of Akt, p-Akt, Bcl-xL and Bcl-2 in HepG2 cells overexpressing DCAF1 transfected with PARD3-shRNA were detected by Western blot. G The protein levels of Akt, p-Akt, Bcl-xL and Bcl-2 in Hep3B cells transfected with DCAF1-shRNA overexpressing PARD3 were detected by Western blot

Fig. 6

DCAF1 regulates HCC cell proliferation and metastasis by activating the Akt signaling pathway. A The protein levels of Akt and p-Akt in Hep3B and SMMC-7721 cells with or without Akt activator were assessed by Western blot. B, D CCK8 assays B, Colony formation assays C and EdU assays D indicated that the Akt activator could partly rescue the repressive effect caused by DCAF1 knockdown on cell proliferation in Hep3B cells (Bar = 100μm (D)). E, F Transwell assays E and wound-healing assays F indicated that the Akt activator partly enhanced HCC cell migration and invasion ability in Hep3B cells transfected with DCAF1-shRNA (Bar = 100μm (E), Bar = 500μm (F)). Data are shown as the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Fig. 7

Combined application of DCAF1 knockdown and Akt inhibitor synergistically suppresses tumor growth. A PARD3 and DCAF1 in tissue chips from HCC patients (n = 20) were detected by IHC (Bar = 100μm). B Hep3B cells with DCAF1 knockdown, Akt inhibitor treatment, or a combination of both were transplanted on nude mice. Tumor volumes and tumor weights were recorded. C Detection of Ki67, DCAF1, PARD3 and p-Akt in the tumor tissues by IHC staining (Bar = 100μm). D Assessment of Akt, p-Akt and EMT marker proteins in xenografts by Western blot. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001