USP1 promotes cholangiocarcinoma progression by deubiquitinating PARP1 to prevent its proteasomal degradation

Abstract

Despite its involvement in various cancers, the function of the deubiquitinase USP1 (ubiquitin-specific protease 1) is unexplored in cholangiocarcinoma (CCA). In this study, we provide evidence that USP1 promotes CCA progression through the stabilization of Poly (ADP-ribose) polymerase 1 (PARP1), consistent with the observation that both USP1 and PARP1 are upregulated in human CCA. Proteomics and ubiquitylome analysis of USP1-overexpressing CCA cells nominated PARP1 as a top USP1 substrate. Indeed, their direct interaction was validated by a series of immunofluorescence, co-immunoprecipitation (CO-IP), and GST pull-down assays, and their interaction regions were identified using deletion mutants. Mechanistically, USP1 removes the ubiquitin chain at K197 of PARP1 to prevent its proteasomal degradation, with the consequent PARP1 stabilization being necessary and sufficient to promote the growth and metastasis of CCA in vitro and in vivo. Additionally, we identified the acetyltransferase GCN5 as acetylating USP1 at K130, enhancing the affinity between USP1 and PARP1 and further increasing PARP1 protein stabilization. Finally, both USP1 and PARP1 are significantly associated with poor survival in CCA patients. These findings describe PARP1 as a novel deubiquitination target of USP1 and a potential therapeutic target for CCA.

Fig. 1. USP1 interacts with PARP1.

A The expression of USP1 in CCA in TCGA databasae. B Proteomics and mass spectrometry analysis of ubiquitination were conducted in RBE cells with stable USP1 overexpression, resulting in the identification of proteins exhibiting reduced ubiquitination levels and increased expression. IgG was used as the isotype control for CO-IP assay. C CO-IP assay was conducted to identify the interaction between USP1 and the 10 candidate proteins identified in B in HuCC-T1. WCL: whole cell lysates. D, F Cell lysates from HuCC-T1 (D), HCCC-9810 (E), and RBE (F) were analyzed by IP using antibodies against USP1 and PARP1, then subjected to IB analysis, respectively. IgG was used as the isotype control. G, H HA-USP1 and Myc-PARP1 were co-transfected into HEK-293T cells. The cell lysates were subjected to IP with anti-HA or anti-Myc antibodies followed by IB analysis with antibodies against Myc and HA. I HuCC-T1, HCCC-9810, and RBE cells were fixed and stained with USP1 antibody (Green) and PARP1 antibody (Red). Nuclei were stained with DAPI (blue). Scale bar: 10 μm. J After fixation, in situ PLA for USP1/PARP1 was performed with anti-USP1 and anti-PARP1 antibodies. The PLA-detected proximity (PROX) complexes are represented by the fluorescent rolling circle products (red dots). Scale bars, 10 mm. K Purified Myc-PARP1 was incubated with GST, GST-USP1, or GST-USP1 C90S coupled to glutathione-Sepharose beads. Proteins retained on Sepharose were then subjected to IB analysis with indicated antibodies. Recombinant GST-USP1 and GST-USP1 C90S was purified from bacteria and analyzed by SDS-PAGE and Coomassie blue staining. L, M Schematic representation of HA-tagged full-length (FL) USP1, Myc-tagged FL PARP1, and their various deletion mutants. N, O HEK-293T cells were co-transfected with Myc-PARP1 and HA-tagged FL USP1 or its deletion mutants, and cell lysates were analyzed by IP with HA (N) or Myc (O) beads followed by IB analysis with antibodies against Myc and HA. P HEK-293T cells were co-transfected with HA-USP1 and Myc-tagged FL PARP1 or its deletion mutants, and cell lysates were analyzed by IP with HA beads followed by IB analysis with antibodies against HA and Myc.

Fig. 2. USP1 enhances the stability of PARP1.

A Increasing amounts of USP1 were transfected into HEK-293T cells and PARP1 expression was detected by IB analysis. B, C HuCC-T1 and HCCC-9810 cells transfected with two independent USP1 shRNA were treated with or without the proteasome inhibitor MG132 (20 μM, 8 hours) and autophagy inhibitor CQ (25 μM, 2 hours), and then USP1 and PARP1 were analyzed. D Detection of PARP1 protein level by IB analysis after treatment with DMSO or proteasome inhibitor MG132 (20 μM) for 8 hours in RBE transfected with vector or USP1. E, F The qRT-PCR was used to detect the mRNA level of USP1 and PARP1 in HuCC-T1 (E) and HCCC-9810 (F) cell lines transfected with the indicated shRNAs. G, H IB analysis was conducted to assess PARP1 protein levels in HuCC-T1 and HCCC-9810 cells with stable USP1 knockdown, combined with overexpression of either HA-USP1 or HA-USP1 C90S, respectively. I IB analysis of PARP1 was conducted in RBE cells overexpressing USP1 or with combined knockdown of USP1. J, K RBE cells were transfected with USP1 or USP1 C90S, treated with 50 μg/ml CHX, collected at the indicated times, and then subjected to IB analysis with antibodies against PARP1 and USP1. Quantification of PARP1 levels relative to β-actin is shown (K). L–O HuCC-T1 (L, M) and HCCC-9810 (N, O) cells stably expressing control shRNA or two independent USP1 shRNA were treated with 50 μg/ml CHX, harvested at the indicated times, and then subjected to IB analysis with antibodies against PARP1 and USP1. Quantification of PARP1 levels relative to β-actin is shown. The data were obtained from three independent biological replicates and are presented as mean ± SD. One-way ANOVA with Dunnett’s post test (E, F, K, M, O). ***p < 0.001, **p < 0.005, ^#p > 0.05.

Fig. 3. USP1 removes the K48-linked ubiquitin chain from K197 of PARP1.

A HuCC-T1 and HCCC-9810 were co-transfected with the indicated ShRNA and His-Ub, and cell lysates were subjected to IP with PARP1 antibody, followed by IB analysis with antibodies against His and PARP1. Cells were treated with 20 μM MG132 for 8 hours before harvesting. B HEK-293T or RBE cells were co-transfected with Myc-PARP1, His-Ub, and HA-USP1 or USP1 C90S, and cell lysates were subjected to IP with Myc beads followed by IB analysis with antibodies against His and Myc. Cells were treated with 20 μM MG132 for 8 hours before harvesting. C HEK-293T cells was co-transfected with Myc-PARP1, His-Ub, and Increasing amounts of HA-USP1, and cell lysates were subjected to IP with Myc beads followed by IB analysis with antibodies against His and Myc. Cells were treated with 20 μM MG132 for 8 hours before harvesting. D Ubiquitylated Myc-PARP1 was incubated with purified HA-USP1 or HA-USP1 C90S from HEK-293T cells. Detection of Myc-PARP1 and His-Ub was performed by IB analysis using anti-Myc and anti-His antibodies, respectively. The purity of the purified HA-USP1 and HA-USP1 C90S proteins was assessed by SDS-PAGE followed by Coomassie blue staining. E Ubiquitination mass spectrometry of the PARP1 peptide in RBE cell with overexpressed USP1. F HEK-293T cells were co-transfected with HA-USP1, His-Ub, and Myc-PARP1 or Myc-PARP1-K197R, and cell lysates were subjected to IP with Myc beads followed by IB analysis with antibodies against His and Myc. Cells were treated with 20 μM MG132 for 8 hours before harvesting. G HEK-293T cells were co-transfected with Myc-PARP1, HA-USP1, and the indicated His-Ub WT, or His-K48-Ub (Lys48-only), or His-K63-Ub (Lys63-only) plasmids, and then the PARP1 ubiquitylation linkage was analyzed by IB analysis using anti-His antibody. H HEK-293T cells were co-transfected with Myc-PARP1, HA-USP1, and the indicated His-Ub WT, or His-K6-Ub (Lys6-only), or His-K11-Ub (Lys11-only), or His-K27-Ub (Lys27-only), or His-K29-Ub (Lys29-only), or His-K33-Ub (Lys33-only) plasmids, and then the PARP1 ubiquitylation linkage was analyzed by IB analysis using anti-His antibody. I Purified HA-USP1 was incubated with K48-linked di-ubiquitin in the presence of the compound at 37 °C for 1.5 hours. Samples were then analyzed by IB analysis using an anti-Ub antibody.

Fig. 4. USP1 promotes CCA proliferation, invasion, and metastasis through PARP1.

A–C Colony assay was assessed in HuCC-T1 transfected with shCtrl, USP1-sh1, or USP1-sh2, reconstituted with PARP1. Additionally, colony assay was conducted on RBE cells transfected with either vector or USP1 and reconstituted with PARP1 sh1. Representative images are presented in A, while the relative number of clones is quantified in B, C. D, E The Transwell assay was performed on HCCC-9810 cells transfected with shCtrl, USP1-sh1 or USP1-sh2, and reconstituted with PARP1. Representative images are presented in E, while the relative number of migrated cells was quantified D. F, G The Transwell assay was performed on RBE cells transfected with either vector or USP1 and reconstituted with PARP1 sh1. Representative images are presented in F, while the relative number of migrated cells was quantified in G. H–J The Xenograft tumor formation assay was performed on HuCC-T1 cells transfected with shCtrl, USP1-sh1, and reconstituted with PARP1. The indicated cells were subcutaneously injected (2.5 × 10⁶ cells per mouse) into nude mice (n = 6). Gross images of the tumors were as shown in H. The growth curve of the tumor is shown in I. The weight of the tumors was shown in J. K–M The Xenograft tumor formation assay was performed on RBE cells transfected with either vector or USP1 and reconstituted with PARP1 sh1. The indicated cells were subcutaneously injected (2.5 × 10⁶ cells per mouse) into nude mice (n = 6). Gross images of the tumors were as shown in K. The growth curve of the tumor was shown in L. The weight of the tumors was shown in M. N–O The lung metastasis model assay was performed on HuCC-T1 cells transfected with shCtrl, USP1-sh1, and reconstituted with PARP1. Representative images are shown in N, and the relative number of lung metastases is shown in O. P, Q The lung metastasis model assay was performed on RBE cells transfected with either vector or USP1 and reconstituted with PARP1 sh1. Representative images are shown in P, and the relative number of lung metastases is shown in Q. Data are from three independent biological repeats and presented as mean ± SD. ***P < 0.001, **p < 0.005, one-way ANOVA with Dunnett’s post test.

Fig. 5. GCN5 induces acetylation of USP1 to strengthen PARP1 stability.

A Sequence alignment of the conserved K130 containing region in USP1 orthologs of different species. B Nicotinamide (NAM) (5 mM, 4 hours) and trichostatin A (TSA) (0.5 μM, 16 hours) were used to improve protein acetylation levels, and acetylated USP1 in HuCC-T1 and HCCC-9810 was IP with pan-acetylation (Pan-AC) antibody followed by IB analysis with antibodies against USP1. C In HEK-293T cells, HA-USP1 was co-expressed with Flag-tagged P300, GCN5, PCAF, CBP, or Tip60 acetyltransferases respectively. The cell lysates were subjected to IP with anti-Flag followed by IB analysis with antibodies against HA and Flag. D Cell lysates from HuCC-T1 cells were analyzed by IP using antibodies against USP1 and GCN5, then subjected to IB analysis. E HuCC-T1, HCCC-9810, and RBE cells were fixed and stained with USP1 antibody (Green) and GCN5 antibody (Red). Nuclei were stained with DAPI (blue). Scale bar: 10 μm. F HA-USP1 WT and Flag-GCN5 were co-expressed in HEK-293T cells with or without MB-3. The cell lysates were subjected to IP with anti-HA followed by IB analysis with antibodies against HA, Pan-AC, and Flag. G Flag-GCN5 and HA-USP1 WT or HA-USP1 K130R mutants were co-overexpressed in HEK-293T cells. The cell lysates were subjected to IP with anti-HA followed by IB analysis with antibodies against HA, Pan-AC, and Flag. H Myc-PARP1 and HA-USP1 or HA-USP1 K130Q mutants were co-overexpressed in HEK-293T and RBE cells. The cell lysates were subjected to IP with anti-HA followed by IB analysis with antibodies against HA and Myc. I IB analysis of Myc-PARP1 was conducted in RBE and HEK-293T cells overexpressing HA-USP1 or HA-USP1 K130Q. J IB analysis of total lysates and anti-Myc immunoprecipitates of HEK-293T cells transfected with His-Ub, HA-USP1/K130R, or Myc-PARP1 constructs and treated with MG132 (20 μM for 8 hours) as indicated. K IB analysis of total lysates and anti-Myc immunoprecipitates of HEK-293T cells transfected with His-Ub, HA-USP1/K130Q, or Myc-PARP1 constructs and treated with MG132 (20 μM for 8 hours) as indicated. L IB analysis of total lysates and anti-Myc IP of HEK-293T and RBE cells transfected with His-Ub, HA-USP1/K130R, Flag-GCN5 or Myc-PARP1 constructs and treated with MG132 (20 μM for 8 hours) as indicated. M, N RBE cells were transfected with USP1 or USP1 K130Q, treated with 50 μg/ml CHX, collected at the indicated times, and then subjected to IB analysis with antibodies against PARP1 and USP1. Quantification of PARP1 levels relative to β-actin is shown (M). O, P With or without Flag-GCN5, RBE cells were transfected with either USP1 WT or USP1 K130Q, treated with 50 μg/ml CHX, collected at designated time points, and subsequently subjected to IB analysis using antibodies against PARP1 and USP1. The quantification of PARP1 levels relative to β-actin is presented. The data were obtained from three independent biological replicates and are presented as mean ± SD. One-way ANOVA with Dunnett’s post test (M, O). ***P < 0.001, *p < 0.05.

Fig. 6. USP1 is enriched in CCA tissues and its expression correlates negatively with patients’ survivals.

A IB analysis of cell lysates from 28 CCA samples using USP1 and PARP1 antibodies. B Correlation analysis of USP1 and PARP1 in CCA samples (n = 28). Chi-square test was used for statistical analysis. Pearson r represents the correlation coefficient. C Representative images of IHC staining of USP1 and PARP1 on tissue of CCA patients (n = 65). Scale bars are indicated. D Correlation analysis of IHC-scores of USP1 and PARP1 in CCA samples (n = 65). Chi-square test was used for statistical analysis. Pearson r represents the correlation coefficient. E Kaplan–Meier curves showing overall survival of CCA patients divided based on USP1 expression in tumors (P values by log-rank test). F Kaplan-Meier curves showing overall survival of CCA patients divided based on PARP1 expression in tumors (P values by log-rank test). G The graphic summary created by Adobe Illustrator, depicts the regulation of PARP1 by USP1. Specifically, GCN5 acetylates USP1 at K130 to enhance its affinity for PARP1. Additionally, USP1 stabilizes PARP1 protein by removing the ubiquitin chain at K197 and promotes CCA progression.