背景:紫杉醇(PTX)治疗耐药是导致三阴性乳腺癌(TNBC)预后不良的重要因素,因此,迫切需要确定联合治疗的新靶点。Neddylation是一种翻译后过程,它引入了一种称为神经前体细胞的泛素样蛋白,该蛋白在发育中表达下调蛋白8(NEDD8)。以前的研究已经发现,在多种肿瘤中,neddylation被激活,但其与PTX化疗敏感性的关系尚未见报道。
方法:使用公共数据库和免疫组织化学验证了PTX敏感和PTX不敏感的TNBC组织之间UBC12和NEDD8表达水平的差异。通过体外和体内功能实验观察Neddylation抑制联合PTX治疗对肿瘤进展的影响。共同IP,采用westernblot和PCR方法研究其分子机制。分子对接用于模拟UBC12和TRIM25的蛋白质结合。分子动力学模拟观察TRIM25蛋白构象的变化。
结果:我们发现在对PTX不敏感的TNBC中,NEDD8和NEDD8接合酶UBC12高度表达。用NEDD8激活酶(NAE)抑制剂mln4924或UBC12敲低治疗显着增加了肿瘤对PTX的敏感性,这种敏感性的增加与UBC12介导的自噬激活有关。机械上,UBC12可以在K117处将NEDD8转移到含有25(TRIM25)的E3泛素连接酶三方基序。分子动力学模拟表明,TRIM25的neddylation修饰降低了其RING域的空间位阻,促进TRIM25和泛素化底物的结合。随后,TRIM25通过增加转录因子EB(TFEB)的K63-聚泛素化促进自噬相关基因的核易位和转录,从而降低肿瘤对PTX的敏感性。
结论:Neddylation在PTX不敏感的TNBC中被激活。具体来说,UBC12/TRIM25/TFEB轴介导的自噬基因转录激活降低了TNBC对PTX的敏感性。Neddylation抑制与PTX治疗的组合显示出协同抗肿瘤作用。
BACKGROUND: Paclitaxel (PTX) treatment resistance is an important factor leading to poor prognosis in triple-negative breast cancer (TNBC), therefore there is an urgent need to identify new target for combination therapy.
Neddylation is a post-translational process that introduces a ubiquitin-like protein called neural precursor cell expressed developmentally downregulated protein 8 (NEDD8). Previous studies have found that
neddylation is activated in multiple tumors, but its relationship with PTX chemotherapy sensitivity has not been reported.
METHODS: Differences in UBC12 and NEDD8 expression levels between PTX-sensitive and PTX-insensitive TNBC tissues were validated using public databases and immunohistochemistry. The in vitro and in vivo functional experiments were used to observe the effect of
neddylation inhibition combined with PTX therapy on tumor progression. Co-IP, western blot and PCR assays were used to investigate the molecular mechanisms. Molecular docking was used to simulate the protein binding of UBC12 and TRIM25. Molecular dynamics simulation was used to observe the changes in TRIM25 protein conformation.
RESULTS: We found that in TNBC that is insensitive to PTX, NEDD8 and NEDD8 conjugating enzyme UBC12 are highly expressed. Treatment with the NEDD8-activating enzyme (NAE) inhibitor mln4924 or knockdown of UBC12 significantly increased the sensitivity of the tumor to PTX, and this increase in sensitivity is related to UBC12-mediated autophagy activation. Mechanistically, UBC12 can transfer NEDD8 to E3 ubiquitin ligase tripartite motif containing 25 (TRIM25) at K117. Molecular dynamics simulations indicate that the neddylation modification of TRIM25 reduces steric hindrance in its RING domain, facilitating the binding of TRIM25 and ubiquitylated substrates. Subsequently, TRIM25 promotes the nuclear translocation of transcription factor EB (TFEB) and transcription of autophagy related genes by increasing K63-polyubiquitination of TFEB, thereby reducing tumor sensitivity to PTX.
CONCLUSIONS: Neddylation is activated in PTX-insensitive TNBC. Specifically, autophagy gene transcriptional activation mediated by the UBC12/TRIM25/TFEB axis reduces TNBC sensitivity to PTX.
Neddylation suppression combination with PTX treatment shows a synergistic anti-tumor effect.