UBE2C-induced crosstalk between mono- and polyubiquitination of SNAT2 promotes lymphatic metastasis in bladder cancer
Wenjie Li, Changhao Chen, Hanhao Zheng, Yan Lin, Mingjie An, Daiyin Liu, Yonghai Zhang, Mingchao Gao, Tianhang Lan, Wang He
Wenjie Li, Changhao Chen, Hanhao Zheng, Yan Lin, Mingjie An, Daiyin Liu, Yonghai Zhang, Mingchao Gao, Tianhang Lan, Wang He
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Research Article Cell biology Oncology

UBE2C-induced crosstalk between mono- and polyubiquitination of SNAT2 promotes lymphatic metastasis in bladder cancer

Abstract

Ubiquitination plays an essential role in protein stability, subcellular localization, and interactions. Crosstalk between different types of ubiquitination results in distinct biological outcomes for proteins. However, the role of ubiquitination-related crosstalk in lymph node (LN) metastasis and the key regulatory factors controlling this process have not been determined. Using high-throughput sequencing, we found that ubiquitin-conjugating enzyme E2 C (UBE2C) was overexpressed in bladder cancer (BCa) and was strongly associated with an unfavorable prognosis. Overexpression of UBE2C increased BCa lymphangiogenesis and promoted LN metastasis both in vitro and in vivo. Mechanistically, UBE2C mediated sodium-coupled neutral amino acid transporter 2 (SNAT2) monoubiquitination at lysine 59 to inhibit K63-linked polyubiquitination at lysine 33 of SNAT2. Crosstalk between monoubiquitination and K63-linked polyubiquitination increased SNAT2 membrane protein levels by suppressing epsin 1–mediated (EPN1-mediated) endocytosis. SNAT2 facilitated glutamine uptake and metabolism to promote VEGFC secretion, ultimately leading to lymphangiogenesis and LN metastasis in patients with BCa. Importantly, inhibition of UBE2C significantly attenuated BCa lymphangiogenesis in a patient-derived xenograft model. Our results reveal the mechanism by which UBE2C mediates crosstalk between the monoubiquitination and K63-linked polyubiquitination of SNAT2 to promote BCa metastasis and identify UBE2C as a promising target for treating LN-metastatic BCa.

Authors

Wenjie Li, Changhao Chen, Hanhao Zheng, Yan Lin, Mingjie An, Daiyin Liu, Yonghai Zhang, Mingchao Gao, Tianhang Lan, Wang He

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Figure 4

UBE2C increases the membrane expression level of SNAT2 by inhibiting its endocytosis.

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UBE2C increases the membrane expression level of SNAT2 by inhibiting its...
(A) IF assays showing the localization of SNAT2 after overexpression of UBE2C. Scale bar: 5 μm. Original magnification, ×4 (enlarged insets). (B) FACS analysis and quantification of SNAT2 expression in the membrane after overexpression of UBE2C. (C) IB analysis of SNAT2 expression in membrane fractions after overexpression of UBE2C. (D) IF analysis of the localization of SNAT2 in BCa cells subjected to cold blockade. Scale bar: 5 μm. Original magnification, ×4 (enlarged insets). (E) IB analysis of SNAT2 expression in membrane fractions after overexpression of UBE2C in cold blocks. (F) IB analysis of SNAT2 expression in membrane and endosome fractions after overexpression of UBE2C and SNAT2K59R mutation. (G) IF assays showing the colocalization of SNAT2 and RAS oncogene family member (RAB5) after overexpression of UBE2C and the SNAT2K59R mutation. Scale bar: 5 μm. (H) MS analysis for the detection of SNAT2-interacting proteins. (I) IB analysis of the interaction between SNAT2 and EPN1 after overexpression or knockdown of UBE2C. (J and K) IB (J) and FACS (K) analysis of SNAT2 expression in membrane fractions after overexpression of EPN1 and UBE2C. (L) IF assays showing the colocalization of SNAT2 and RAB5 after the overexpression of EPN1 and UBE2C. Scale bar: 5 μm. (M) IB analysis of the interaction between SNAT2 and EPN1 after SNAT2K33R mutation. (N) IB analysis of the interaction between SNAT2 and EPN1 after SNAT2K59R mutation. Significant differences were identified through 2-tailed Student’s t test (B) and 1-way ANOVA followed by Dunnett’s test (K). Quantitative results are presented as the mean ± SEM of 3 separate experiments.