USP1 Maintains the Survival of Liver Circulating Tumor Cells by Deubiquitinating and Stabilizing TBLR1

Abstract

The prognosis of hepatocellular carcinoma (HCC) is closely associated with the occurrence of distant metastases, which is likely due to circulating tumor cells (CTCs). However, the low number of CTCs is the main obstacle limiting research of the mechanism of CTC metastasis. Here, We evaluated the role of ubiquitin-specific protease 1 (USP1) in promoting CTC survival during blood-borne metastases. We observed that USP1 was frequently upregulated in CTCs and correlated with metastasis and a reduced overall survival rate of patients. Additionally, genetic knockout of USP1 the survival rate of CTCs. Further analyses showed that USP1 mediates oncogenic activity by deubiquitinating and stabilizing transducin β-like 1 X-linked receptor 1 (TBLR1), which plays essential roles in regulating Wnt signaling. These results demonstrated that USP1 may act as an essential factor in promoting the survival of CTCs and suggest that inhibition of USP1 is a potential strategy for HCC treatment.

Keywords: USP1; Wnt pathway; circulating tumor cells; deubiquitination; hepatocellular carcinoma.

FIGURE 1

Elevated expression of USP1 in HCC. (A) USP1 mRNA levels in 40-paired specimens of HCC tissues and matched para-tumor liver tissues. (B) IHC staining of USP1 in 56 pairs of HCC and matching para-tumor tissues. Representative images are shown. (C) USP1 levels in seven paired samples. T, HCC tissues; P, para-tumor tissues. β-actin was used as a control. (D) Kaplan–Meier analysis with log-rank testing of survival was performed in 217 patients with HCC exhibiting different USP1 expression levels. Error bars represent the standard deviation. *P = 0.025. (E,F) Cell numbers were count by cell counting instrument after 0, 24, 48, and 96 h of seeding.

FIGURE 2

USP1 promotes circulating tumor cell survival in blood. (A) USP1 mRNA expression level in primary tumors and CTCs. (B) USP1 expression at 0, 12, 24, and 36 h after tail injection. (C) After 0, 12, 24, and 36 h of tail injection, GFP-labeled cells were sorted and counted by FACS. (D) 24 h after injection, GFP-labeled cells were sorted and labeled with Annexin V-APC and 7AAD. (E) Intrahepatic metastatic tumors in hepatic lobes 5 weeks after liver xenografting. (F,G) Lung metastasis detected by computed tomography and IHC. The arrows were used to show lung metastasis. (H) Bar graph of pulmonary metastases tumor numbers. (I) CTCs detected in blood and labeled by EpCAM and pan-cytokeratin (green) and DAPI (red). *P < 0.05.

FIGURE 3

USP1 KO impairs Wnt targets. (A) KEGG pathway enrichment of differentially expressed genes (DEGs) in USP1-KO cells versus control cells (in MHCC-97H cell line). (B) GSEA enrichment plots of Wnt signaling and Notch signaling. (C) Identification of USP1-deubiquitinating targets using a combination of ubiquitin-chain-specific IP and label-free LC-MS/MS analysis. (D) Venn diagram showing the number of ubiquitin-modified proteins in the two groups. (E) Venn diagram showing five overlapping proteins between the USP1-KO group and Wnt signaling.

FIGURE 4

USP1 interacts, deubiquitinates, and stabilizes TBLR1. (A) Co-IP assays of USP1 and TBLR1 in MHCC-97H cells. (B) Impact of USP1 on TBLR1 ubiquitination in vivo. Immunoblot using an HA-tag to detect poly-ubiquitination of TBLR1. (C) USP1 enhances TBLR1 stability; cells were treated with CHX (100 μg/mL) and collected at 0, 3, 6, and 9 h. TBLR1 levels were analyzed by western blotting.

FIGURE 5

Overexpressing TBLR1 rescues the survival of CTC by USP1 depletion. (A) TBLR1 overexpression rescued the expression of Wnt targets caused by USP1-knockout. (B,C) Constructed from the MHCC-97H cell line, USP1-NC-GFP-vector cells, USP1-KO-GFP-vector cells, USP1-NC-GFP-TBLR1-OE cells, and USP1-KO-GFP-TBLR1-OE cells were injected into the peripheral tail vein. After 24 h, the CTC number (B) and cell-survival rate (C) was detected by FACS. The CTC number (B) and cell-survival rate (C) of USP1-NC-GFP-vector cells, USP1-KO-GFP-vector cells, USP1-NC-GFP-TBLR1-OE cells, and USP1-KO-GFP-TBLR1-OE cells after 24 h of tail injection. *P < 0.05.

FIGURE 6

The mechanism of USP1 maintaining the survival of CTCs by stabilizing TBLR1.