YY1 induced USP13 transcriptional activation drives the malignant progression of hepatocellular carcinoma by deubiquitinating WWP1

Abstract

Background: Hepatocellular carcinoma (HCC) is the sixth most prevalent cancer globally and the third leading cause of cancer-related mortality. Protein ubiquitination and deubiquitination play vital roles in human cancers. Ubiquitin-specific protease 13 (USP13) is a deubiquitinating enzyme (DUB) that is involved in many cellular processes. However, the mechanism by which USP13 regulates deubiquitination remains largely unknown.

Methods: Clinical data were analyzed via online databases. USP13 expression in HCC cell lines and tissues was analyzed via western blotting and immunohistochemistry. A lentivirus was used to established stable USP13-knockdown and USP13-overexpression cells. Cell Counting Kit-8, colony formation, wound healing, Transwell, and sphere formation assays were used to detect the malignant behaviors of HCC cells in vitro. A subcutaneous mouse model was used to investigate the function of USP13 in vivo. Co-immunoprecipitation, chromatin immunoprecipitation and dual-luciferase reporter assays were conducted to explore the molecular regulation.

Results: USP13 was upregulated in HCC cell lines and tissues, which predicted a poor prognosis in patients with HCC. Functional experiments in which USP13 was overexpressed or depleted revealed the oncogenic role of USP13 in driving HCC progression both in vitro and in vivo. Mechanistically, WW domain-containing ubiquitin E3 ligase 1 (WWP1) was identified as a binding protein of USP13. Furthermore, USP13 can interact with WWP1 and then remove the K29- and K48-linked polyubiquitination chains from WWP1 to stabilize the WWP1 protein via the ubiquitin-proteasome pathway. Moreover, Yin Yang 1 (YY1) was explored as a new transcription factor of USP13, and YY1 could also upregulate WWP1 expression through USP13. Moreover, YY1 and WWP1 were shown to participate in the oncogenic role of USP13.

Conclusions: Our findings revealed the functional YY1/USP13/WWP1 signaling axis in HCC, identifying a promising therapeutic target for anti-HCC treatment.

Keywords: Deubiquitination; HCC; USP13; WWP1; YY1.

Fig. 1

USP13 is highly expressed in HCC and predicts poor prognosis. A USP13 mRNA expression levels in different tumor tissues and normal tissues from the GEO and the TCGA databases were analyzed with Xena (http://xena.ucsc.edu/). The blue bars represent normal tissues and red bars represent tumor tissues. B USP13 mRNA levels in HCC and normal liver tissue was analyzed based on TCGA. C USP13 mRNA levels in HCC and normal liver tissue was analyzed based on the GEO (GSE54236) database. D Representative IHC staining images of USP13 in HCC and adjacent tissues. E Quantitative analysis of IHC staining results by ImageJ. F–H Prognostic information including overall survival (OS), disease-specific survival (DSS), and progression-free interval (PFI) were analyzed in the TCGA database. I USP13 mRNA levels in different HCC cell lines. J, K USP13 protein levels in different HCC cell lines, and quantified by ImageJ. Scale bars: 50 µm

Fig. 2

USP13 was associated with HCC cell proliferation, migration, and stemness. A, B Huh7 and MHCC97H were infected with shNC, shUSP13-1, and shUSP13-2 lentivirus. qRT-PCR and WB were used to determine the mRNA and protein level of USP13. C, D CCK-8 and (E) colony formation assays were used to detect cell proliferation ability. F Transwell and (G) wound healing assays were used to detect cell migrate ability. H Spheroid formation assay was used to detect cell stemness. I Several stemness-related proteins expression was detected. Scale bars: 275 µm

Fig. 3

USP13 interacted with WWP1 in HCC cells. A Co-IP assay detected the endogenous interaction between USP13 and WWP1 with a USP13 antibody in HCC cells and HEK293T cells. B Co-IP results with a WWP1 antibody in HCC cells and HEK293T cells. C Co-IP assays showed the exogenous interaction between USP13 and WWP1 with a Flag antibody. D Co-IP results of the binding between USP13 and WWP1 with a Myc antibody. E The interaction between USP13 truncation mutants and WWP1. F Immunofluorescence indicated the colocalization of USP13 and WWP1 in Huh7 and MHCC97H cells. Scale bars: 50 µm

Fig. 4

USP13 removed the K29- and K48-linked polyubiquitination chain of WWP1. A WB results of WWP1 expression upon USP13 knockdown in HCC cells. B WB results of WWP1 expression upon USP13 overexpression in HCC cells. C, D The ubiquitination of WWP1 was analyzed in HCC cells with stable USP13 knockdown or overexpression. E WWP1 protein level was detected in HCC cells treated with CHX and MG132 or HCQ. F CHX assays were conducted to detect the half-life of WWP1 in Huh7 shUSP13 and shNC cells. G Quantitative analysis of WWP1 protein expression by ImageJ. H, I WB results of the ubiquitination of WWP1 in HEK293T cells with co-transfection of Myc-WWP1, HA-ubiquitin, or mutations, and either the empty vectors or Flag-USP13

Fig. 5

USP13 regulates HCC cell behaviors via WWP1. A WB assay detected the Akt/mTOR pathway-related protein expression levels with WWP1 overexpression in USP13-knockdown HCC cells. B WB assay detected the Akt/mTOR pathway-related protein expression levels with WWP1-knockdown in USP13-overexpression HCC cells. C CCK-8 and (D) colony formation assays were used to detect cell proliferation ability. E Spheroid formation assays were used to detect cell stemness ability. F, G Wound healing and (H, I) Transwell assays were used to detect cell migration ability. Scale bars: 275 µm

Fig. 6

USP13 regulated tumorigenesis of HCC cells via WWP1 in vivo. A The nude mice bearing tumors were euthanized and photographed. B Images of tumors from the nude mice in A. A ruler was used to indicate the size of tumors. C Tumor growth curves from the nude mice in A . D The nude mice bearing tumors were euthanized and photographed. E Images of tumors from the nude mice in D. A ruler was used to indicate the size of tumors. F Tumor growth curves from the nude mice in D . G, I IHC analysis of Ki67 expression in tumors shown in B and E. H, J Quantification and statistical analysis of the Ki67 IHC scores. K, L USP13 and WWP1 protein level in tumors from nude mice were detected by WB. Scale bars: 50 µm

Fig. 7

YY1 transcriptionally activates USP13 and forms a carcinogenic YY1/USP13 axis in HCC. A The transcriptional factors that regulate USP13 were predicted by PROMO, AliBaba2.1, Animal TFDB, and GeneCards. B, C WB and qRT-PCR results of USP13 expression after knockdown or overexpression of YY1. D ChIP-qPCR analyzed the binding motifs of USP13 promoter with YY1 protein in MHCC97H cells. E The binding motif 3 of the USP13 promoter was mutated, and the relative luciferase activity from wild-type and mutant-type-transfected cells was measured by a dual luciferase reporter system. F WWP1 proteins were detected by WB with overexpression of USP13 in YY1-knockdown HCC cells. G WWP1 proteins were detected by WB with knockdown of USP13 in YY1-overexpression HCC cells. H Representative IHC staining image of YY1, USP13 and WWP1 in HCC tissue were displayed. I The correlation between YY1 and USP13 were analyzed based on IHC score. J The correlation between USP13 and WWP1 was analyzed on the basis of IHC score. K The correlation of between YY1 and WWP1 was analyzed according to IHC score. Scale bars: 50 µm

Fig. 8

YY1 regulates HCC cell proliferation and migration via USP13. A CCK-8 assay of YY1-knockdown in USP13-overexpression and YY1-overexpression in USP13-knockdown HCC cells were used to detect cell proliferation ability. B, C Colony formation assays confirmed the difference in cell proliferation. D, E Wound healing assays were used to detect cell migration ability of the above cells. Scale bars: 275 µm

Fig. 9

The mechanistic scheme of this study