USP9X expression was downregulated in cholangiocarcinoma patients and correlated with better prognosis
To determine the functional and clinical relevance of genes in cholangiocarcinoma, we first analyzed the genes that affect overall survival (OS) in cholangiocarcinoma from TCGA using GEPIA online website (http://gepia2.cancer-pku.cn). Among these screened genes in the datasets, that patients with high USP9X expression had better OS (Fig. 1A). To validate this finding, we investigated the effect of USP9X expression on postoperative survival in cholangiocarcinoma patients by immunohistochemistry (IHC). Univariate statistical methods were utilized to analyze clinical data from 54 cholangiocarcinoma patients. Patients were allocated to two groups based on the IHC score of USP9X, namely, high-expression group (USP9X > 8, n = 29) and low-expression group (USP9X ≤ 8, n = 25). OS was significantly different between the two groups. The results demonstrated that high expression levels of USP9X are associated with better prognosis of patients with cholangiocarcinoma in OS (Fig. 1B). Representative IHC images are shown in Fig. 1C. These results suggested that the USP9X gene might play a central role in cholangiocarcinoma progression. Furthermore, we evaluated the expression level of USP9X in a cohort of 6 pairs of cholangiocarcinoma and para-tumor (non-cancerous) tissues by immunoblotting. The results suggested that compared with cholangiocarcinoma tissues, USP9X expression level was significantly higher in para-tumor tissues (Fig. 1D). IHC analysis for 54 cases of cholangiocarcinoma tissues and 12 cases of para-tumor tissues also showed that positive staining of the USP9X protein was enriched in para-tumor tissues, but was rarely observed in cholangiocarcinoma tissues (Fig. 1E and 1F). These results suggested that the USP9X gene might play a suppressive role in cholangiocarcinoma progression.
USP9X suppresses cholangiocarcinoma cells proliferation both in vitro and in vivo
To investigate the function of USP9X in the progression of cholangiocarcinoma, RBE and HUCCT human cholangiocarcinoma cell lines were overexpressed USP9X (Fig. 2A and 2B). Cell proliferation assays using CCK-8 kit revealed that overexpression of USP9X inhibited cell proliferation in RBE (Fig. 2C) and HUCCT cells (Fig. 2D). Colony growth assays also indicated that expression of USP9X decreased colony formation of RBE cells (Fig. 1E and 1F). The same inhibition for colony formation was also observed in USP9X overexpression HUCCT cells (Fig. 1G and 1H). To evaluate whether the expression level of USP9X could affect cholangiocarcinoma cell growth in vivo, We also established stable USP9X knockdown HUCCT cholangiocarcinoma cells with USP9X shRNA (Supplementary Figure S1A). We subcutaneously injected USP9X knockdown or control USP9X cells into nude mice. The mice were euthanized, compared to control cells, the proliferation rate was significantly increased in USP9X knockdown HUCCT cells (Fig. 2I). The subcutaneous tumors were measured every 0.5 weeks until 5.5 weeks after cell injection (Fig. 2J). The tumor weight was significantly increased in the USP9X-silenced groups than in the control groups (Fig. 2K). Furthermore, IHC staining of 54 cases of cholangiocarcinoma tissue suggested that the expression of Ki-67 were markedly downregulated in USP9X high samples (Fig. 2L) but upregulated in USP9X low samples, which demonstrated that the expression level of USP9X was negatively correlated with Ki67 expression.
USP9X interacts with EGLN3 and promotes its expression
To further characterize the regulatory effect of USP9X on cell proliferation. An integrated bioinformatics platform (BioGRID) for investigating the protein interaction network (https://thebiogrid.org) was utilized to predict the substrate of USP9X. We noticed that EGLN3 may serve as a binding partner for USP9X. To achieve this, we next examined whether USP9X interacts with EGLN3. The interaction between USP9X and EGLN3 at the endogenous protein levels was validated in RBE and HUCCT cells by co-immunoprecipitation with an anti- USP9X antibody and anti-EGLN3 (Fig. 3A). Reciprocal IP assays demonstrated that USP9X and EGLN3 formed a ternary complex at the exogenous level in HEK293T cells (Fig. 3B). On the basis of these analyses, we next examined whether EGLN3 expression correlates with USP9X in cholangiocarcinoma cells. We observed that knockdown of USP9X in RBE and HUCCT cells was responsible for the decreased expression level of EGLN3 (Fig. 3C and 3D). On the contrary, the expression level of EGLN3 was significantly increased after overexpression of USP9X in RBE and HUCCT cells (Fig. 3F and 3G). Those data indicate that USP9X may play a positive regulatory role on expression of EGLN3.
USP9X regulates EGLN3 at protein level.
To pursue the regulation of EGLN3 by USP9X, we co-overexpress wild-type USP9X and EGLN3 in HEK293T cells. We observed that only wild‐type USP9X but not its catalytically inactive mutant (C1566S) upregulated the protein levels of exogenously expressed EGLN3 in HEK293T (Fig. 4A). Compared with wild‐type USP9X, catalytically inactive mutant (C1566S) was also deprived of ability for promoting the protein levels of endogenously expressed EGLN3 in RBE cells (Fig. 4B). Similarly, inhibition of USP9X by a partially selective inhibitor WP113046 impaired promotion of EGLN3 by USP9X in RBE cells (Fig. 4C). Those data have been shown that USP9X promotes EGLN3 protein level in a catalytic activity dependent manner. Then, mRNA of EGLN3 were examined by RT‐qPCR analysis. Results showed that USP9X depletion by two respective siRNAs did not affect mRNA levels of EGLN3 but significantly reduced its protein levels in HUCCT and RBE cells (Fig. 4D). The increased USP9X also have no influence in mRNA levels of EGLN3 (Fig. 4E). Moreover, the addition of WP113046 made no difference to mRNA levels of EGLN3 (Fig. 4F). USP9X-mediated deregulation of EGLN3 in RBE and HUCCT cells were effectively restored after treatment with 10 µM of proteasome inhibitor MG-132 for 6 h (Fig. 4G). These results indicate the regulation of EGLN3 by USP9X to be posttranscriptional.
USP9X enhances the stability of EGLN3 and counteracts its ubiquitination
To test whether USP9X regulates EGLN3 protein stability, RBE and HUCCT cells stably expressing shNC, shUSP9X #1 and shUSP9X #2 were treated with 100 µg/mL CHX. Samples were collected at the indicated times and then subjected to immunoblotting analysis with the indicated antibodies. As shown in Fig. 5A, B, the half-life of EGLN3 in cells expressing shUSP9X #1 and shUSP9X # was significantly shorter than that in cells expressing shNC, indicating that USP9X enhances the stability of EGLN3 protein. Inhibition of USP9X by WP113046 also reduced stability of EGLN3 protein (Fig. 5C and 5D). On the contrary, overexpression of USP9X enhanced half-life of EGLN3 (Fig. 5E and 5F). As USP9X is a substrate-specific deubiquitinase, we next examined the effect of USP9X knockdown on EGLN3 ubiquitination. Toward this aim, RBE cells were transfected with shNC and shUSP9X. After 48 hours of transfection, cells were treated with 10 µmol/L MG-132 for 6 hours and then total cellular lysates were subjected to IP assays with anti-EGLN3 antibody. Immunoblotting analysis showed that USP9X knockdown significantly increased the ubiquitination of EGLN3 protein (Fig. 5G). In agreement with these observations, overexpression of USP9X in RBE cells decreased the ubiquitination levels of endogenous EGLN3 (Fig. 5H). Moreover, addition of WP113046 showed a significant increase of polyubiquitinated exogenous DIAPH3 protein in HEK293T cells.
USP9X inhibited growth of cholangiocarcinoma through EGLN3
To further identify the clinical relevance of our findings, we evaluated the correlation of expression levels between USP9X and EGLN3 in 5 pairs of primary cholangiocarcinoma and matched adjacent noncancerous tissues by immunoblotting. As mentioned above, the protein level of USP9X in cholangiocarcinoma tissues was lower than adjacent noncancerous tissues (Fig. 6A). Moreover, the level of EGLN3 in cholangiocarcinoma tissues was higher than adjacent noncancerous tissues (Fig. 6A). These results verified the positive relationship of expression level between USP9X and EGLN3. EGLN3 was documented to inhibit cells proliferation and metastasis in other tumors including prostate cancer, glioma, pancreatic cancer and breast cancer26,31,32. But, the function of EGLN3 for cholangiocarcinoma remains unknown. Therefore, we examined the function of EGLN3 in cholangiocarcinoma. The results demonstrated that engraft tumors expressing shEGLN3 grew faster than those expressing empty vector (Fig. 6B-6D). To explore whether tumor growth promoted by USP9X is due to deubiquitinate EGLN3, cell proliferation assays using colony growth assays revealed that decreased cell proliferation caused by overexpression of USP9X rescued by knockdown of EGLN3 (Fig. 6E-6F).
USP9X positively regulated the expression level of apoptosis pathway genes
To elaborate the regulatory role of USP9X on the cell proliferation, we further explore the downstream of USP9X and EGLN3. EGLN3 has been reported to promote the expression of KIF1Bβ involved in apoptosis24. Frequent deletions of the kinesin-like protein gene 1B (KIF1B) have been reported in neural tumors33. Recently, a genome-wide association study demonstrated an association between polymorphisms in the KIF1B gene and the risk of hepatocellular carcinoma (HCC)24. Another study documented that downregulation of KIF1B mRNA in hepatocellular carcinoma tissues correlates with poor prognosis34. Genetic variations in KIF1B also are reported to contribute to risk of epithelial ovarian cancer (EOC)35. These studies suggested that KIF1B may play an important role in the development of other tumors besides nerve tumors. We next validated the regulatory effect of USP9X on components of the apoptosis signaling pathway. Western blot analysis showed that KIF1Bβ and another apoptosis signaling pathway components, as well as apoptosis signaling marker, c-casp3, were increased in USP9X overexpression cells compared to pCDH cells (Fig. 7A). Additionally, shUSP9X treatment resulted in significant decrease of expression level of KIF1Bβ and c-casp3 (Fig. 7B). The increased expression level of KIF1Bβ and c-casp3 promoted by overexpressed USP9X impaired by knockdown of EGLN3 using two respective shRNAs (Fig. 7C). These results suggested that USP9X play a regulatory role on apoptosis signaling pathway through its substrate EGLN3. To further confirm the regulatory role of USP9X on apoptosis signaling, Flow cytometry was utilized to detect apoptosis of RBE cells. Strong positive correlations were observed between USP9X expression and cells apoptosis (Fig. 7D and 7E). Similarly, the increased apoptosis rate induced by overexpression of USP9X was weaken by knockdown of EGLN3 (Fig. 7D and 7E). Morphological changes in apoptotic cells provide essential markers for defining and detection of apoptosis as a fundamental mechanism of cell death. Among these changes, the nuclear fragmentation and condensation have been regarded as the important markers. We observed that the rates of nuclear fragmentation and condensation for RBE cells increased significantly after overexpression of USP9X. On this basis, EGLN3 was knockdown using respective shRNAs. As expected, the increased rates of nuclear fragmentation and condensation was eliminated and as much as RBE cells expressing pCDH (Fig. 7F).
In conclusion, our results unveil USP9X as a potential diagnostic, prognostic and therapeutic tool for cholangiocarcinoma, which provides a novel approach for use in noninvasive screening of cholangiocarcinoma. Findings presented here showed that USP9X plays a suppressive role in cholangiocarcinoma progression (Fig. 8). USP9X exerts its tumorigenic suppressive functions through deubquitin-dependent of EGLN3. USP9X functions as a tumor suppressor to participate in apoptosis activation of cholangiocarcinoma. Upregulated DIAPH3 increased the protein level of KIF1Bβ and markers of apoptosis, c-casp3. The suppressive regulation of USP9X in tumor is potentially important. Consequently, evaluating the therapeutic potential of USP9X in cholangiocarcinoma deserve large-scale studies.