In July 2023, the article "UBE2S interacting with TRIM21 mediates the K11-linked ubiquitination of LPP to promote the lymphatic metastasis of bladder cancer" published in Cell Death & Disease(IF 9.0), this article uses perrotine cell-free protein expression kit to synthesize ubiquitin-K11R protein, and deeply explores the mechanism of ubiquitination in bladder cancer in lymphatic metastasis. In this paper, cell-free expression technology is used to achieve more efficient, rapid and flexible synthesis of recombinant proteins, which greatly reduces the experimental cycle and labor costs.
 

research background

Bladder cancer (BCa) is one of the most common malignancies in the genitourinary system, with 573,000 newly diagnosed cases and 213,000 deaths reported worldwide in 2020. Lymphatic metastasis is the most common mode of bladder cancer (BCa) metastasis, and the prognosis is very poor. Although some new urinalysis and therapeutic agents have been used, muscle invasive BCa(MIBC) has a poor prognosis. The expression of ubiquitin conjugating enzyme E2S(UBE2S) has been reported to be up-regulated in all types of cancer and is associated with poor prognosis. However, the biological mechanism of UBE2S in BCa lymphatic metastasis remains to be discovered.

The present study was designed to illustrate the interaction of UBE2S with TRIM21 (a member of the ternary motif family) and to find that degradation of LPP (lipomatous chaperone) by K11-associated ubiquitination promotes lymphatic metastasis of BCa. UBE2S was demonstrated to be a potent therapeutic target for metastatic BCa. Queue Settings

Cohort 1: included 210 BCa tissue specimens and 59 normal urothelial tissues;
Cohort 2: consisting of 59 paired microarray tissues;
Oomics data: TCGA and GEO(GSE48277,GSE120736 and GSE31684);
In addition, the authors downloaded Ubiquitination-related genes (KEGG UBIQUITIN MEDIATED PROTEOLYSIS) from MSigDB.

Research Results

1.UBE2S was positively correlated with lymph node metastasis and poor prognosis of BCa
The researchers performed a bioinformatic analysis of the TCGA-BLCA database and identified key ubiquitinases in BCa lymphatic metastasis. In addition, UBE2S was confirmed to be a highly expressed gene associated with ubiquitination and metastasis in TCGA-BLCA by differential expression analysis. Subsequently, using IHC detection, the researchers detected UBE2S protein levels in Cohort 1, found that UBE2S was localized in the nucleus and cytoplasm, but little in NAT, and showed a gradual increasing trend from non-muscle invasive bladder cancer (NMIBC) to muscle invasive bladder cancer (MIBC), MIBC without lymphatic metastasis (LN-MIBC) to lymphatic metastatic MIBC(LN MIBC). The high expression rate of UBE2S was consistent in BCa tissues compared to NAT in the TCGA database (FIG. 1E). The researchers compared different cohorts and database information (Figure 1F-P) and performed Kaplan-Meier survival (Figure 1Q-1R), and found that high expression of UBE2S is a risk factor for OS and DFS in BCA patients. The results showed that the high expression of UBE2S was not only related to lymph node metastasis, advanced tumor and histological grade, but also predicted the poor prognosis of BCA patients.
 

Figure 1:UBE2S is positively associated with lymphatic metastasis and poor prognosis in bladder cancer (BCa)

2.UBE2S knockdown inhibits BCa migration and invasion in vitro and lymphatic metastasis in vivo
The authors constructed stable UBE2S-knockdown and UBE2S-overexpressing models using T24 and UM-UC-3 cell lines. Wound healing and transwell migration analysis showed that the migration speed and the number of BCa cells in the UBE2S knockdown group were greatly reduced (Figure 2B,C), and the invasion ability of BCa cells was significantly inhibited when UBE2S was silenced (Figure 2D), but it was enhanced when UBE2S was overexpressed. An in vivo popliteal LN model was established using stable UBE2S-knockdown UM-UC-3 cells (FIG. 2E), and a significant decrease in luminescence of politeal LNs was observed in the UBE2S-knockdown group compared to the control group. UBE2S silencing also significantly reduced the volume of popliteal LNs. At the same time, H & E staining based on Popliteal LNs showed that the percentage of lymph node metastasis in the UBE2S knockdown group was reduced from 100 in the matched group to 20% (Figure 2J,K). In conclusion, UBE2S increased BCa cell migration and invasion in vitro and lymphatic metastasis in vivo.

Figure 2:UBE2S knockdown inhibits BCa migration and invasion in vitro and lymphatic metastasis in vivo

3.UBE2S interacts with TRIM21 and LPP in the nucleus and cytoplasm of BCa
The researchers carried out immunoprecipitation and mass spectrometry analysis of wild-type T24 and UM-UC-3 cells with UBE2S antibodies, and found 10 E3 enzymes, of which TRIM21, UBR5 and UBE3C were reported to be associated with tumor metastasis. In addition, three additional metastasis-associated genes (TRIP13, LPP, and PRMT1) were selected for further studies on 27 potential substrate proteins of UBE2S. Finally, the interaction between UBE2S, TRIM21 and LPP was confirmed by co-IP and western blot analysis (Figure 3A), and further immunofluorescence analysis showed that UBE2S, TRIM21 and LPP were highly co-localized in BCa nucleus and cytoplasm (Figure 3B). Furthermore, LPP protein expression was increased in UBE2S-knockdown BCa cells but decreased in UBE2S-overexpressing cells, but TRIM21 expression was not affected (FIG. 3C). TRIM21 knockdown increased LPP protein expression levels (FIG. 3D). Subsequent qPCR analysis found that knockdown of UBE2S and TRIM21 did not significantly increase the mRNA level of LPP, indicating that UBE2S and TRIM21 regulate LPP mainly through post-translational modification.

Figure 3:UBE2S interacts with TRIM21 and LPP in the nucleus as well as in the cytoplasm of BCa.

4.UBE2S interacts with TRIM21 to degrade LPP through K11 linked ubiquitination
The differences in LPP protein levels were significantly attenuated after treatment with MG132, a proteasome inhibitor, indicating that UBE2S mediated LPP degradation through ubiquitination. Next, the researchers performed in vivo ubiquitination analysis in HEK293T cells, and in addition, the ubiquitin-K11R protein was synthesized using the Perotene cell-free protein synthesis kit for in vitro ubiquitination analysis. The results indicate that UBE2S, in combination with TRIM21, mediates the formation of K11 links on LPP. Further, the authors constructed a TRIM21 plasmid lacking the loop domain (ΔRING) (FIG. 4I), confirming that the RING domain of TRIM21 is required for LPP ubiquitination.
 

Figure 4:UBE2S interacts with TRIM21 to degrade LPP by inducing K11-linked ubiquitination

5.LPP inhibits the transfer effect of UBE2S on BCa
The researchers performed IHC tests in cohort 2, and a significant negative correlation was observed between the expression of UBE2S and LPP (Figure 5A,B); the chi-square test showed that LPP expression was negatively correlated with N and M disease stages. And LPP expression showed a trend of sequential adjustment from NAT, LN-CA to LN CA (Figure 5C). Kaplan-Meier survival analysis of cohort 2 found that BCa patients with low LPP expression had a poor prognosis and lower OS and DFS rates (Figure 5E,F). In addition, both univariate and multivariate analyses indicated that low LPP expression was a risk factor for OS and DFS in BCA patients. Then, it was investigated whether UBE2S exerts oncogenic function in an LPP-dependent manner by transfection of siRNA (FIG. 5G). The results showed that LPP silencing significantly increased migration and invasion of BCa cells (Figure 5H-J). Thus, LPP acts as a tumor suppressor in BCa and reverses the metastasis-promoting effect of UBE2S.

Figure 5:LPP reverses the transfer-promoting effect of UBE2S

6.UBE2S promotes EMT of BCa cells in a LPP-dependent manner

Next, the authors examined the expression of EMT markers by western blot. The results indicate that UBE2S enhances the metastasis of BCa cells by promoting the EMT process (FIG. 6A). In addition, LPP knockdown increased protein levels of mesenchymal markers but decreased epithelial marker protein levels in BCa cells (FIG. 6B). Immunofluorescence staining showed that silencing UBE2S attenuated EMT, while overexpressing UBE2S accelerated EMT (FIG. 6C). In addition, EMT markers were detected in tumor tissues derived from UBE2S-knockdown in vivo model using IHC analysis (FIG. 6D). After inhibition of LPP, UBE2S knockdown significantly restored the inhibition of EMT (6E). These data demonstrate that UBE2S promotes EMT in BCa cells in an LPP-dependent manner.

Figure 6:UBE2S promotes EMT in BCa cells in an LPP-dependent manner

7.Cephalomannine (cephalotaxine) pharmacologically inhibits UBE2S and prevents lymphatic metastasis of BCa cells

The authors then explored the functional role of Cephalomannine, a small molecule compound that inhibits the activity of the UBE2S promoter. In BCa cells treated with Cephalomannine, the expression of UBE2S was gradually decreased in a dose-dependent manner (FIG. 7A). Transwell migration and invasion analysis showed that Cephalomannine had a strong inhibitory effect on BCa cells (Fig. 7B,C). The model of lymphatic metastasis showed a significant decrease in both relative luminescence and popliteal LN volume in a dose-dependent manner for Cephalomannine treatment group (Figure 7D, H). In addition, IHC examination of tumor tissues showed that Cephalomannine decreased the expression of UBE2S, N-cadherin, vimentin and Ki-67, but increased the expression of LPP and N-cadherin in a concentration-dependent manner (FIG. 7K). Blood analysis revealed no apparent toxicity of the Cephalomannine. These data demonstrate that Cephalomannine inhibits BCa cell metastasis both in vitro and in vivo by targeting UBE2S.

Figure 7:Cephalomannine pharmacologically inhibits UBE2S and blocks lymphatic metastasis of BCa cells

Cephalomannine Inhibits Growth and Metastasis of Human BCa Derived Organoids
To delve into the clinical value of Cephalomannine in BCa, the authors established four patient-derived organoids (PDOs)(FIG. 8A). Functionally, it was shown that Cephalomannine interfered with the growth and proliferation of BCa organoids in a dose-dependent manner (FIG. 8B,C). And BCa organoids were significantly more sensitive to Cephalomannine (FIG. 8D). In addition, Cephalomannine was also found to reduce the expression of the mesenchymal marker N-cadherin, indicating that Cephalomannine blocked EMT in BCa organoids (FIG. 8E).

Figure 7:Cephalomannine inhibits growth and metastasis of organoids from human BCa

Conclusion
This study identified a novel biomarker and therapeutic target, UBE2S. Providing evidence for the critical role of UBE2S in BCa lymphatic metastasis, the UBE2S-TRIM21-LPP axis was then investigated as a novel proteasome-mediated mechanism, and UBE2S was found to interact directly with TRIM21. The Ubiquitin-K11R protein was synthesized using the Perotin cell-free protein expression kit to further study the mechanism of ubiquitination, and it was determined that UBE2S-induced LPP down-regulation occurred in a ubiquitin-proteasome-dependent manner. K11-linked Cephalomannine enhances the metastatic capacity and EMT of BCa cells by degrading LPP, and inhibits the proliferation and metastasis of human BCa-derived organoids. There is no apparent toxicity.

 

Published literature
Xiao K, Peng S, Lu J, Zhou T, Hong X, Chen S, Liu G, Li H, Huang J, Chen X, Lin T. UBE2S interacting with TRIM21 mediates the K11-linked ubiquitination of LPP to promote the lymphatic metastasis of bladder cancer. Cell Death Dis. 2023 Jul 8;14(7):408. doi: 10.1038/s41419-023-05938-2. PMID: 37422473; PMCID: PMC10329682.