USP9X promotes apoptosis in cholangiocarcinoma by modulation expression of KIF1Bβ via deubiquitinating EGLN3

Abstract

Background Cholangiocarcinoma represents the second most common primary liver malignancy. The incidence rate has constantly increased over the last decades. Cholangiocarcinoma silent nature limits early diagnosis and prevents efficient treatment. Methods Immunoblotting and immunohistochemistry were used to assess the expression profiling of USP9X and EGLN3 in cholangiocarcinoma patients. ShRNA was used to silence gene expression. Cell apoptosis, cell cycle, CCK8, clone formation, shRNA interference and xenograft mouse model were used to explore biological function of USP9X and EGLN3. The underlying molecular mechanism of USP9X in cholangiocarcinoma was determined by immunoblotting, co-immunoprecipitation and quantitative real time PCR (qPCR). Results Here we demonstrated that USP9X is downregulated in cholangiocarcinoma which contributes to tumorigenesis. The expression of USP9X in cholangiocarcinoma inhibited cell proliferation and colony formation in vitro as well as xenograft tumorigenicity in vivo. Clinical data demonstrated that expression levels of USP9X were positively correlated with favorable clinical outcomes. Mechanistic investigations further indicated that USP9X was involved in the deubiquitination of EGLN3, a member of 2-oxoglutarate and iron-dependent dioxygenases. USP9X elicited tumor suppressor role by preventing degradation of EGLN3. Importantly, knockdown of EGLN3 impaired USP9X-mediated suppression of proliferation. USP9X positively regulated the expression level of apoptosis pathway genes de through EGLN3 thus involved in apoptosis of cholangiocarcinoma. Conclusion These findings help to understand that USP9X alleviates the malignant potential of cholangiocarcinoma through upregulation of EGLN3. Consequently, we provide novel insight into that USP9X is a potential biomarker or serves as a therapeutic or diagnostic target for cholangiocarcinoma.

TGFBR2 is a novel substrate and indirect transcription target of deubiquitylase USP9X in granulosa cells

“…In mouse ovary, Usp9x is enriched in the GCs of primordial and primary follicles, which indicates that Usp9x is related to mammalian folliculogenesis (Sato et al, 2004). Herein, we demonstrated that USP9X is an antiapoptotic posttranslational modifier, which inhibits the (Chen et al, 2021;Vucic et al, 2011). Multiple components such as c-FLIP and ASK1 in the death receptor pathway, and MCL-1 of the mitochondrial pathway are direct substrates for DUB USP9X (Kim et al, 2019;Lai et al, 2021;Nagai et al, 2009;Schwickart et al, 2010).…”

“…Multiple components such as c‐FLIP and ASK1 in the death receptor pathway, and MCL‐1 of the mitochondrial pathway are direct substrates for DUB USP9X (Kim et al, 2019; Lai et al, 2021; Nagai et al, 2009; Schwickart et al, 2010). Additionally, USP9X has an indirect impact on cell apoptosis via the cell death signaling pathways: for example, USP9X induces apoptosis in cholangiocarcinoma cells by increasing the expression of cleaved‐caspase3, a common effector of both the death receptor and the mitochondrial pathways through deubiquitination and preventing the degradation of EGLN3 (Chen et al, 2021). In summary, our study demonstrates for the first time that USP9X is essential for GC function and follicular development in female mammals.…”

“…Herein, we demonstrated that USP9X is an antiapoptotic posttranslational modifier, which inhibits the apoptosis of porcine GCs. Increasing evidence from functional assays has demonstrated that USP9X acts as a vital modulator of cell apoptosis via direct or indirect regulation of the cell death‐related pathways including the extrinsic death receptor pathway and intrinsic mitochondrial pathway (Chen et al, 2021; Vucic et al, 2011). Multiple components such as c‐FLIP and ASK1 in the death receptor pathway, and MCL‐1 of the mitochondrial pathway are direct substrates for DUB USP9X (Kim et al, 2019; Lai et al, 2021; Nagai et al, 2009; Schwickart et al, 2010).…”

USP9X deubiquitinates and stabilizes CDC123 to promote breast carcinogenesis through regulating cell cycle

“…In our previous studies, we found that the physical association of USP9X with centrosomes was dependent on the cell cycle and mainly occurred in the S and G2 phases in breast cancer 32 . Another study showed that USP9X plays an anticancer role by stabilizing EGLN3, whose overexpression can arrest cancer cells in the G1 phase and mediate cell apoptosis 49 . In human neural progenitor cell lines, USP9X was found to stabilize RAPTOR protein levels and loss of USP9X resulted in cell cycle arrest in the G0 phase and decreased mTORC1 signaling, which is a major regulator of G0/G1 cell cycle progression 50 .…”

USP9x promotes CD8 <sup>+</sup> T-cell dysfunction in association with autophagy inhibition in septic liver injury

“…USP9X was reported to be an oncogene that promotes tumorigenesis and chemoresistance by regulating tumor cell apoptosis [26] and participating in the activation of TCR signaling. USP9x was found to be indispensable for lymphocyte activation, either by controlling ZAP70 ubiquitination or modulating the carma1-bcl10-malt1 complex, but the molecular mechanisms are not clear [ 12 ‒ 14 ] .…”