Ubiquitin-specific protease 7 is a drug-able target that promotes hepatocellular carcinoma and chemoresistance

The HCC fresh tissue from HCC patients (Affiliated hospital of Nantong University). The liver cell (LO2) and HCC cells (SMMC-7721, SK-Hep1, HepG2, Huh7) were purchased from ATCC (USA). LO2 was cultured in 1640 DMEM + 10% FBS. HCC cells were cultured in DMEM + 10% FBS. All cells lines were cultured in 37 °C with 5% CO2 concentration.

The paraffin-embedded tissues sections from 100 matched pairs of liver adenocarcinoma and nonmalignant liver tissue of the same patients. The tissues were heated at 60 °C for 45 min and deparaffinized using a graded ethanol series. Then, these tissue sections were incubated in sodium citrate buffer and heated for antigen retrieval and endogenous peroxidase activity was blocked by soaking in 0.3% hydrogen peroxide. USP7 (Santa Cruz, CA, USA) was incubated for 1 h at room temperature. The second antibody was then incubated. All sections were counterstained with hematoxylin, dehydrated and evaluated. Staining of USP7 in the tissues was reviewed and scored independently by two pathologists blinded to the clinical data. The intensity of immunostaining was documented as 0–3 (0, negative; 1, weak; 2, moderate; 3, strong). The percentage of immunoreactive cells was documented as 1 (0–25%), 2 (26–50%), 3 (51–75%), and 4 (76–100%). Under these conditions, the optimum cut-off value of samples were respectively classified as low and high expression USP7 by receiver operating curve (ROC) analysis. The area under the curve (AUCs) at different cut-off values of USP7 for different years of overall survival time were calculated.

The USP7 inhibitor P22077 and Doxorubicin (Dox) was purchased from Selleckchem (Houston, TX, USA), dissolved and diluted in dimethyl sulfoxide (DMSO; Sigma, St. Louis, MO, USA), and stored at − 20 °C, DOX was keep from light. The antibodies were used in Western Blot: anti-USP7 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-H3, anti-H3K4me², anti-Bax, anti-GAPDH (Cell Signaling Technology, Inc, Beverly, MA, USA).

The whole cell lysates come from the cell which were treatment with USP7 inhibitor for 48 h at the indicated doses, and was measured using western blot analysis. The protein were subjected to SDS-PAGE and transferred to PVDF membranes (New England Nuclear, Boston, MA,USA). Subsequently,the PVDF was membranes was blocked with 5% nonfat dry milk with TBS-T which containing Tris-buffered saline and 0.1% Tween-20. Probed with the primary antibodies. After washing with them TBS-T buffer, the cell lysates were incubated with the secondary antibody. The chemiluminescence system (Bio-Rad, USA) was used to detect the protein expression.

We designed two sgRNA to knock out the potential sequence of USP7. The vector plasmids were digested by BSPQI enzyme (New England Biolads) at 50 °C for one hour. The sense and antisense sgRNA oligos targeting the predicted super-enhancer locus were annealed at 90 °C for 20 min followed by cooling at room temperature for an hour. The annealing buffer was as following: 10 mM Tris (pH 7.5), 1 mM EDTA and 50 mM NaCl. The ligation was executed with T4 DNA ligase (New England Biolads) at 16 °C overnight. All plasmids were validated by using enzyme digestion and direct DNA sequencing.

Place the cells into a 6-well plate at the appropriate density. After the cells adhere to the cells within 24 h, transfection is performed. Lipofectamine 2000 was diluted in DMEM medium at various concentrations to find the optimal transfection dose per cell. Mix appropriate amounts of DNA and Lipofectamine 2000 with a little DMEM, respectively, and incubate for 5 min at room temperature. Finally, the DNA was mixed with Lipofectamine 2000 and incubated for 15 min, then added to the cell culture medium, and the medium was changed 6 h after transfection. Due to the high transfection efficiency of the HEK293T cell line, all of our SgRNA transfection was performed first in HEK293T cells. After confirming its knockout in HEK293T cells, stable cell lines with corresponding knockouts will be established in tumor cells.

To construct stable USP7 knockout cell line, SK-hep1 cells and Huh7 cells were transfected by SgUSP7-1, SgUSP7-2 plasmids and transfected by epi-CRISPR vector plasmid as control. After 48 h of transfection, the medium was changed by complete medium containing 1 μg/ml puromycin (InvivoGen). Puromycin-containing medium was further used for additional 2 weeks to select the resistant cells until the stable cell line was established, and always use a concentration of 0.5 μg/ml puromycin to maintain.

Suitable cells were in 24-well plate treated with P22077 for 12 h, subsequently, cells treated with Dox at indicated doses for 48 h. Cells were fixed by 4% Paraformaldehyde after the cells rinsed by PBS for 3 times. Cells were closed with Triton X-100 for 30 min and then using anti-USP7 antibody at overnight. After washing with the PBS, the cells were stained with fluorescent dyes (Themorfer, USA) for 2 h and take photos from the light.

The cell proliferation and viability were detected by the cell counting kit-8 (CCK-8; Best Bio, Shanghai, China) assay. Cells were inoculated into 96-well plates at a density of 2 × 10⁴/well and cultivated for 24 h. Each well incubated with CCK-8 reagents for another at the same time on consecutive days. Last, the automated plate reader was used to read the absorbance at a wavelength of 450 nm.

Cells were treated with P22077 or Dox for 48 h were prepared for the apoptosis detection assay. Cells were washed in PBS at least three times, resuspended in 195 μl of binding buffer and incubated with Annexin V-fluorescein isothiocyanate (Best Bio, Shanghai, China) for 15 min in the dark. By followed, cells were incubated with PI (Best Bio, Shanghai, China) in the dark for 5 min, then analysed by flow cytometry (Beckman Coulter Inc., USA) processes.

500–1000 cells in a six-well plate were incubated for 24 h and then p22077 were added to stimulating the cells for 2 weeks. Colonies which were formed macroscopic, fixed by 4% Paraformaldehyde for 2 h and the purple crystal were used to color. The colonies were counted and calculated compared with the control group.

The Scratch assays was used to examined the migration of the cell treated with P22077 at indicated doses for 24 h or 48 h. Subconfluent cells of each group were scraped using sterilized 10 µl pipette tips, washed with PBS, and cultured in six-plated. Wound healing was observed under the microscope (USA) and capture the images.

Four week old male nude mice were purchased from Nantong University Animal Center (Nantong, China) and the SK-Hep1 cell were injected in specific pathogen-free conditions for 5 × 10⁶ cells into left flank of the nude mice. The nude mice were divided into two groups contained the control group and the experimental group with three mice. P22077 was injected into mice at the dose of 10 mg/kg by intraperitoneal injection of the experimental group. All mice were sacrificed after 6 weeks. Tumors were removed, weighed, and fixed in formalin for the subsequent analysis.

All statistical analyses were carried out using GraphPad version 5.0 (Graph Pad software, USA). Significance was analyzed using SPSS 15.0 and student’s t-test. All data are shown as means and standard errors of the mean. P < 0.05 was considered indicate a statistically significant.

USP7 acts as a deubiquitinating enzyme not only contributes to tumorigenesis but also plays critical role in response to therapy. It becomes one of most interesting drug-able targets in cancers but its role as well as potential therapeutic value in HCC remains unclear. We firstly explored USP7 expression using WB in 8 paired HCC tissues. In majority of cases, USP7 was overexpressed in most of HCC tissues than adjacent tissues (Fig. 1a). To further confirm this finding, we examined the expression and distribution of USP7 in HCC tissues by IHC. As showed in Fig. 1b, adjacent liver tissues showed negative staining or slight positive staining, while HCC tissues possess large amount of USP7 and USP7 predominantly locates in the nucleus (Fig. 1b). In the cohort of 100 HCC patients, USP7 expression is highly correlated with unfavorable clinical outcomes (Fig. 1c). Together, our results clearly demonstrate that USP7 is correlated with patient outcome. To further explore the role of USP7 in HCC development, we analyzed which factors are associated with the level of USP7. We divided HCC patients into different groups based on the IHC scores of USP7. As shown in Table 1, high USP7 level was associated with tumor size (P = 0.018), tumor differentiation (P = 0.007), advanced TNM stage (P = 0.004) and liver cirrhosis (P = 0.025). These results indicates that USP7 plays a critical role in the development of HCC and its expression is associated with patients’ outcome.

Due to the high expression of USP7 in HCC and the correlation between USP7 and patients outcome, we examined whether HCC cells relies USP7 for survival. The USP7 inhibitor P22077 was used to treat a panel of HCC cell lines (Huh7, HepG2, SK-Hep1, SMMC-7721) and control cells (LO2). After treatment for 24 h, the viability of Huh7 and SK-Hep1 is significantly reduced under the dose of 10 or 20 µM. Under the same condition, P22077 has minimal effect on control cells (LO2) as well as HCC cells (HepG2, SMMC-7721) (Fig. 2a). Increased treatment time further induces cell death in HuH7 cells and SK-Hep1 cells (Fig. 2b). The same results were also confirmed in USP7 deficient stable cell lines including HuH7 and SK-Hep1 in Additional file 1: Figure S1c. Flowcytometry analysis demonstrated that P22077 treatment induces apoptosis in Huh7 and SK-Hep1 cells but not in SMMC-7721 cells (Fig. 2c). We chose SK-Hep1 USP7 stable cells to reach the same conclusion (Additional file 1: Figure S1e). Microscopic images of cell morphology further showed that P22077 dramatically induced cell death in Huh7 and SK-Hep1 cells but not in SMMC-7721 cells (Fig. 2d). Consistently, we found that the function of USP7 is essential of partial of HCC cells.

We further verify the potential anti-tumor ability of P22077 on HCC cells, Huh7 and SK-Hep1 were treated with P22077 at a concentration of 5 µM, 10 µM and 20 µM. As shown in Fig. 3a, the number of cell foci were obviously decreased in lower dose, the higher dose almost completely abolished the anchor dependent colony-forming ability of these two cells. The similar result was shown in Fig. 2b, P22077 drastically reduced ability of anchor-independent colony formation in Huh7 and SK-Hep1 (Fig. 3b). Subsequently, in vitro scratch assays were undertaken. Compared with DMSO group, Huh7 and SK-Hep1 exhibited delayed wound healing after treated with P22077 for 24 h or 48 h (Fig. 3c). Colony formation, anchor-independent colony formation and scratch assays are also verified in USP7 deficient stable cells including SK-Hep1 and Huh7 (Additional file 1: Figure S1 a, b, f). Taken together, USP7 is required for HCC colony formation and migration.

Next, we examined whether P22077 could inhibits the growth of HCC in vivo. Hepatoma cells SK-Hep1 were injected subcutaneously in nude mice with concentration of 5 × 10⁶/cell. We divided the nude mice into two groups and treated them with DMSO and P22077, respectively. P22077 was injected intraperitoneally at 10 mg/kg for once every 2 weeks. All mice were sacrificed after 6 weeks, the tumors were weighted and photographed. Compared with DMSO-treated group, P22077 significantly inhibited the tumor growth (Fig. 4a, b). The weight of tumor mass in P22077-treated group was significantly decreased compared to control group (Fig. 4c). Our results revealed that P22077 is a potential anti-tumor drug for significantly inhibiting tumor growth.

By considering the potential application of P22077 in HCC therapy, we tested whether P22077 enhances the killing effect of chemotherapy. Previous experiments have shown that the tested concentration of P22077 has no significant effect on SMMC-7721 and HepG2 cells (Fig. 5a, b). We tested whether P22077 can increase the toxicity of Dox in SMMC-7721 and HepG2. Interestingly, SMMC-7721 and HepG2 are resistant to Dox in our tested doses. However, in the presence of P22077, SMMC-7721 and HepG2 cells are responsive to Dox and show a dose-dependent death. Doxorubicin has inherent fluorescence and thus can be visualized in cells. Consistent with cellular toxicity, the number of Dox-positive cells was significantly increased in the combination with P22077 (Fig. 5c). It suggests that P22077 might help Dox to enter nucleus. To test this hypothesis, we monitored the distribution of Dox in a course of P22077 treatment. As shown in Fig. 5d, Dox early enter into nucleus with the help of P22077. Taken together, our results indicate critical role of USP7 in cancer development and chemoresistance.

Our results indicate that USP7 is a potential target for HCC treatment. To systematically understand the molecular mechanisms, we used mass spectrometry to globally analyze the protein change in response to P22077 treatment. In total, we identified 224 of significant changed proteins. Seventy-one of them are down-regulated while 153 of them are up-regulated (data not shown). Further analysis reveals that down-regulated proteins are enriched in several essential biological processes such as translational initiation, protein targeting to membrane, viral transcription, non-sense-mediated RNA decay (Fig. 6a). Up-regulated proteins are enriched in cell–cell adhesion, protein folding, protein transport, signal transduction and cell proliferation (Fig. 6b). This results suggest that USP7 is involved in many essential pathways in HCC. By looking at each down-regulated proteins, we interestingly found histone H3 is one of most affected protein after P22077 treatment. Due to the critical role of histone H3 in epigenetic regulation of transcription, we further confirmed the result obtained from mass spectrometry by WB. As shown in Fig. 6d, P22077 treatment slightly reduced the level of histone H3 in Huh7 and SK-Hep1 cells. The effect is much potent in the high dose of P22077. P22077 treatment also reduces the H3K4me², which is a marker for transcriptional activation (Fig. 6d). Consistently, the P22077-sensative cell lines Huh7 and SK-Hep1 cells have relative high H3K4me² compared to other tested cells (Fig. 6c). It might be partially explained why Huh7 and SK-Hep1 cells are more dependent on USP7 function. Disruption of the function of USP7 in Huh7 and SK-Hep1 cells greatly induces the expression of BAX, which is a potent inducer for apoptosis (Fig. 6d). Thus, P22077 treatment globally affects the biological processes in HCC and subsequent disruption of essential biological processes further induces BAX-mediated apoptosis.