OTUB1 was upregulated in prostate cancer
We filtered cell cycle progression and in prostate cancer from TCGA database, identified 3480 genes up-regulated and 2592 genes down-regulated, in up-regulated genes, we observed the expression of OTUB1 in PCa is higher than para-carcinoma tissue (Fig 1A). To identify the deubiquitinating enzymes driving the prostate cancer progression, we conducted subsequent experiments and assays.
In order to identify whether clinical data was consistent with the database, we collected clinical prostate cancer tissue and immunohistochemical staining was performed with OTUB1 and ki-67 antibody (Fig 1C, D). The expression of OTUB1 and Ki67 in ADPC and CRPC groups were higher than that in BPH group. The mean optical density values of OTUB1 and Ki67 in ADPC group were higher than BPH, the difference was statistically significant (P<0.05). The concise statistical results were showed that the positive ratios of OTUB1 and Ki67 in ADPC and CRPC groups were higher than BPH group (Fig 1E).
OTUB1 promoted proliferation and invasion of PCa cell.
Wildtype OTUB1 vector was transfected into prostate cancer PC3 and C42 cells, and the expression level of OTUB1 was detected by Western blotting. The cells with significantly increased OTUB1 expression level were obtained for cell functional experiments. The results of Western blotting showed that, compared with the cells in the control group, the expression level of OTUB1 in the cells in the otub1-c91s group (cells introduced by the mutated OTUB1 fragment) was not significantly changed, but the expression level of OTUB1 protein in the otub1 cells was significantly increased (Fig 2A). Image analysis software Image J was used to detect the grayscale value of OTUB1 expression in OTUB1 cells with stable and high expression (Fig 2A), and it was found that the expression level of OTUB1 was more than three times that of OTUB1 C91S cells or control group. For further explanation, we transfected PC3 and C4-2 cells with OTUB1 siRNA, and the expression level of OTUB1 was decreased distinctly compared with si control group(Fig 2B).
Recent studies have shown that OTUB1 is highly expressed in invasive tumor cells and plays an important role in their proliferation and invasion(16-18, 32-34). The migration experiment results of this project showed that in PC3 and C4-2 cells of prostate cancer, the migration and invasion ability of OTUB1 overexpression cells was significantly enhanced compared with control cells (Fig 2C,2E). And the opposite result was found in the PC3 and C4-2 cells transfected with OTUB1 siRNA, the migration and invasion ability of PC3 and C4-2 cells with decreased OTUB1 expression attenuated dramatically (Fig 2D,2F).
Cell scratch method is one of the methods to determine the motion characteristics of tumor cells. Using the experimental model of in vitro cell injury and healing, the tumor cell migration ability was observed by applying scratch injury to monolayer cells cultured in vitro and adding low concentration serum medium to eliminate the interference caused by cell proliferation. An empty scar appeared on the cell layer. After the culture with low concentration serum, the effect of OTUB1 expression level on the migration ability of PC3 and C4-2 cells in prostate cancer was observed. The results showed that, compared with the control group, the migration ability of otub1 overexpression cells was significantly improved (Fig 2G), and the otub1-c91s group has no significant difference with control group. and the difference was statistically significant (P < 0.05). The similar conclusions also were found in the PC3 and C4-2 cells transfected with OTUB1 siRNA, the migration ability of PC3 and C4-2 cells with decreased OTUB1 expression attenuated dramatically (Fig 2H). The results showed that OTUB1 expression could significantly influence the migration ability of the cells (Fig 2G, H).
Effects of increased OTUB1 expression on PC3 and C4-2 cells cycle distribution
Cell growth curves were drawn to detect the proliferation of control, otub1 and otub1 c91s cells at 24h, 48h, 72h and 96h, respectively. After the increase of OTUB1 expression level, the changes of PCa cell growth levels at 24h, 48h, 72h and 96h were observed. The results showed that the growth ability of the cell lines with increased OTUB1 expression was significantly higher than that of control group, while the proliferation ability of otub1 c91s cells with OTUB1 mutation have no significant change with control group (Fig 3A, B). At the same way, we transfected PC3 and C4-2 cells with si otub1, we found the opposite results (Fig 3C, D). Our results showed that OTUB1 expression significantly influenced the proliferation of PC3 and C42 cells (P<0.05) (Fig 3A-D).
The cells are made into a dispersed suspension and inoculated at a low density (2×103cells /chamber). The rate at which a single cell grows, proliferate and forms small groups of cells (clones) is called the clonogenesis rate. All the cells formed obvious colonies after 10 days of culture, and the clonogenesis ability of PC3 and C4-2 cells was significantly enhanced after the increase of OTUB1 expression level, and the difference was statistically significant (P < 0.01) (Fig 3E, F). At the same way, we decrease the expression of OTUB1 via transfecting PC3 and C4-2 cells with si otub1, the clonogenesis ability of PC3 and C4-2 cells also decreased significantly (Fig 3G, H). From above results, we could easily find that the expression level of OTUB1 can influence the ability of forming colonies.
Cell cycle refers to the whole process of continuously dividing cells from the end of the last mitosis to the completion of the next mitosis, including the quiescent phase (G0), early DNA synthesis phase (G1), DNA synthesis phase (S), late DNA synthesis phase (G2), and division phase (M). After the increase of OTUB1 expression level, the proliferation ability of prostate cancer cells was significantly enhanced (P < 0.05) (Fig 3A-D). Cell cycle test results of PC3 and C42 showed that the ratio of G0/G1 phase decreased after OTUB1 expression level increased, while the ratio of G2/M+S phase increased, and the difference was statistically significant (P < 0.05) (Fig 3I,J). Overall, these experiments suggested that OTUB1 may influence PCa cells proliferation through altering cell cycle distribution (Fig 3I, J).
OTUB1 rescue Cyclin E1 from proteasomal degradation
We next investigated how OTUB1 promoted the G1 cell cycle progression, a series of OTUB1-relative proteins were analyzed via geneMANIA online database. Cyclin E1, a cell cycle-relative regulative key protein, was found to interact with OTUB1 closely (Fig 4A). To further explore the relationship between OTUB1 and Cyclin E1, we observed that the expression level of Cyclin E1 augment on the increased OTUB1 group (Fig 4B). We also observed that the protein expression level of OTUB1 and Cyclin E1 after PC3 cell was transfected with OTUB1 siRNA (Fig 4C). From above results, we can speculate that up-regulated OTUB1 could promote the expression of Cyclin E1, we had enough reasons to predict that Cyclin E1 may be regulated by OTUB1.
Further experiments are needed to verify this conclusion. Next, we explored how OTUB1 influence the expression level of Cyclin E1, we treated PC3 cell transfected with negative control siRNA and OTUB1 siRNA with DMSO and 10uM MG132 for 8h respectively. The results demonstrated the stability of Cyclin E1 could be decreased in OTUB1 siRNA transfected group, adding MG132 could preserved the stability partly, showing OTUB1 influenced the expression of Cyclin E1 via a proteasome dependent manner (Fig 4D). To further explained above results, we treated PC3 cell transfected with negative control and OTUB1 siRNA with 10mg/ml cycloheximide (CHX), a protein synthesis inhibitor in eukaryotic cells, in 0h, 4h, 8h and 24h. The results showed that CHX could promote the decease of Cyclin E1 protein, and the decrease rate of Cyclin E1 increased in PC3 cell transfected with OTUB1 siRNA. The detailed decrease rates were showed (Fig 4E, F). Above results implied us that Cyclin E1 might be regulated by OTUB1 via a deubiquitination degration manner, to identify this hypothesis, we applied 10uM MG132 and 200uM chloroquine to PC3 cell transfected with OTUB1 siRNA for 8h. We found that MG132 could retain part stability of Cyclin E1 in PC3 cell transfected with OTUB1 siRNA, and chloroquine not (Fig 4G). The results demonstrated that Cyclin E1 might be degraded in a ubiquitination depend manner instead of lysosomal degradation pathway. To identify Cyclin E1 did be degraded in a ubiquitination depend manner, we conducted an immunoprecipitation assay to determined that OTUB1 could interact with Cyclin E1 (Fig 4H,I). Next, we transfected si OTUB1 in PC3 cell, then another immunoprecipitation assay was conducted to confirm that knockdown OTUB1 could strengthen the intensity of Cyclin E1 ubiquitination (Fig 4J). The results demonstrated that OTUB1 could promote tumor process and proliferation in prostate cancer via stabling the function of Cyclin E1 and increasing its expression level. We analyzed the correlation between OTUB1 and CCNE1 via GEPIA online database, the result presented that OTUB1 was positively related to CCNE1 (R=0.19; P<0.01), the result was displayed in the Figure 4J.
The OTUB1/Cyclin E1 axis promotes prostate cancer cell proliferation
To further explore the influence of OTUB1/ Cyclin E1 upon PCa, we transfected PC3 cell with si Cyclin E1 and otub1 wt. 48h after transfection, the protein expression level of Cyclin E1 was detected, the results demonstrated that otub1 promoted the expression of Cyclin E1 and Cyclin E1 expression was decreased obviously after transfecting si Cyclin E1, interesting that knockdown Cyclin E1 attenuated the effect of otub1 wt increasing the expression of Cyclin E1 (Fig 5A). The proliferation ability was tested by MTT assay, the results demonstrated that knockdown Cyclin E1 obviously postponed proliferation comparing with control transfected group, and the effect of OTUB1 overexpression promoting proliferation was restrained significantly accompany with si Cyclin E1 (Fig 5B). This result was reinforced by healing assay and clone formation experiments, these results were consistent with previous results (Fig 5C, D). These results demonstrated that OTUB1/Cyclin E1 axis could promote proliferation and migration ability in prostate cancer. Disturbing the contact between OTUB1 and Cyclin E1 might serve a potential therapy for prostate cancer.
To identify the possibility of targeting OTUB1/ Cyclin E1 axis treatment in prostate cancer, we treated PC3 cell with RO-3306, a Cyclin E1/CDK2 related inhibitor, in 0, 340nM, 1uM, 2uM, 3Um and 5uM. 24h after treating with RO-3306, we extracted protein and conducted western blotting. We found that the expression of Cyclin E1 was decreased depended on RO-3306 dose. Interesting that RO-3306 also attenuated the effect of otub1 overexpression promoting Cyclin E1 (Fig 5E). More experiments were performed to further explore the influence, the results of MTT and clone formation experiments demonstrated that the proliferation ability would gradually decline with the RO-3306 concentration increasing (Fig 5F, H). And the results of healing assay were consistent with the abovementioned results (Fig 5G). RO-3306 treatment postponed the migration and proliferation ability, the application of RO-3306 to clinical treatment for prostate cancer might be a potential and effective treatment measure, play a role with deceasing the mortality rate of prostate cancer patients.
Increased expression level of OTUB1 promote PC3 cell tumor growth in vivo.
The animal model was male nude mice at 4-6 weeks. We planted 2×106 PC3 cells control and its overexpressed OTUB1 cloned cells otub1 in the groin of mice respectively. When the tumor grew to a diameter of 0.5 cm, we began to measure the tumor size daily with a vernier caliper (Fig 6C). After 12 days, the animals were sacrificed for cervical dislocation, and solid tumor was removed under sterile conditions. The volume of the tumor blocks of otub1 mice overexpressed OTUB1 and control group have distinct difference, and the OTUB1 c91s group has no obvious change with control group (Fig 6A, B). We found that the tumorigenesis rate of OTUB1 overexpressed cells in mice was significantly higher than that of control cells, especially after two weeks of tumorigenesis, which demonstrated the promoting effect of OTUB1 on tumor formation. We observed that the tumor of OTUB1 overexpression group also have high expression of OTUB1, ki-67 and Cyclin E1 with cells experiments compared with control group (Fig 6D).