Cell Biology and Toxicology ( IF 5.3 ) Pub Date : 2022-06-01 , DOI: 10.1007/s10565-022-09729-x Ting Wu 1 , Chengyun Li 1 , Changlong Zhou 1 , Xiaxia Niu 1 , Gege Li 1 , Yali Zhou 2 , Xinsheng Gu 3 , Hongmei Cui 1
|
Background
The mutation of BRAF V600E often occurred in melanoma and results in tumorigenesis. BRAF mutation drives hyperactivation of the RAF-MAPK-ERK pathway. The acquired drug resistance upon prolonged use of BRAF inhibitors (such as vemurafenib) still remains the main obstacle. Previously, we have found that E3 ligase Skp2 over-expresses vemurafenib-resistant melanoma cells, and knockdown of Skp2 enhances the anti-tumor effect of vemurafenib. Interestingly, the literature has reported that the selective USP14/UCHL5 inhibitor b-AP15 displays great potential in melanoma therapy; however, the molecular mechanism still remains unknown.
Methods
In vitro, the effect of the combination regimen of vemurafenib (Vem, PLX4032) and b-AP15 on vem-sensitive and vem-resistant melanoma has been investigated by wound healing, colony formation, transwell invasion assay, flow cytometry, lysosome staining, and ROS detection. In vivo, the combination effect on vem-resistant melanoma has been evaluated with a nude mice xenograft tumor model. GST-pulldown and co-immunoprecipitation (co-IP) assays have been applied to investigate the interactions between USP14, UCHL5, and Skp2. Cycloheximide (CHX) assay and ubiquitination assays have been used to explore the effect of USP14 on Skp2 protein half-life and ubiquitination status.
Results
In the present study, we have revealed that repression of USP14 sensitizes vemurafenib resistance in melanoma through a previously unappreciated mechanism that USP14 but not UCHL5 stabilizes Skp2, blocking its ubiquitination. K119 on Skp2 is required for USP14-mediated deubiquitination and stabilization of Skp2. Furthermore, the mutated catalytic activity amino acid cysteine (C) 114 on USP14 abrogates stabilization of Skp2. Stabilization of Skp2 is required for USP14 to negatively regulate autophagy. The combination regimen of Skp2 inhibitor vemurafenib and USP14/UCHL5 inhibitor b-AP15 dramatically inhibits cell viability, migration, invasion, and colony formation in vemurafenib-sensitive and vemurafenib-resistant melanoma. Vemurafenib and b-AP15 hold cells in the S phase thus leading to apoptosis as well as the formation of the autophagic vacuole in vemurafenib-resistant SKMEL28 cells. The enhanced proliferation effect of USP14 and Skp2 is mainly due to a more effective reduction of cell apoptosis and autophagy. Further evaluation of various protein alterations has revealed that the increased expression of cleaved-PARP, LC3, and decreased Ki67 are more obvious in the combination of vemurafenib and b-AP15 treatment than those in single-drug treatment. Moreover, the co-treatment of vemurafenib and b-AP15 dramatically inhibits the growth of vemurafenib-resistant melanoma xenograft in vivo. Collectively, our findings have demonstrated that the combination of Skp2 inhibitor and USP14 inhibitor provides a new solution for the treatment of BRAF inhibitor resistance melanoma.
中文翻译：
抑制 USP14 通过调节 Skp2 增强对维莫非尼耐药的黑色素瘤的抗肿瘤作用
背景
BRAF V600E突变常发生在黑色素瘤中并导致肿瘤发生。BRAF 突变驱动 RAF-MAPK-ERK 通路过度激活。长期使用 BRAF 抑制剂（如维莫非尼）后获得的耐药性仍然是主要障碍。此前，我们发现E3连接酶Skp2过表达对vemurafenib耐药的黑色素瘤细胞，敲低Skp2可增强vemurafenib的抗肿瘤作用。有趣的是，文献报道选择性USP14/UCHL5抑制剂b-AP15在黑色素瘤治疗中显示出巨大潜力；然而，其分子机制仍不清楚。
方法
在体外，通过伤口愈合、集落形成、Transwell 侵袭试验、流式细胞术、溶酶体染色和ROS检测。在体内，已使用裸鼠异种移植肿瘤模型评估了对 vem 耐药性黑色素瘤的联合作用。GST 下拉和免疫共沉淀 (co-IP) 测定已用于研究 USP14、UCHL5 和 Skp2 之间的相互作用。放线菌酮 (CHX) 测定和泛素化测定已用于探索 USP14 对 Skp2 蛋白半衰期和泛素化状态的影响。
结果
在本研究中，我们发现，USP14的抑制通过一种先前未被认识到的机制使黑色素瘤中的维莫非尼耐药变得敏感，即USP14而不是UCHL5稳定Skp2，阻止其泛素化。USP14 介导的去泛素化和 Skp2 的稳定需要 Skp2 上的 K119。此外，USP14 上突变的催化活性氨基酸半胱氨酸 (C) 114 消除了 Skp2 的稳定性。USP14 需要稳定 Skp2 才能负向调节自噬。Skp2 抑制剂 vemurafenib 和 USP14/UCHL5 抑制剂 b-AP15 的组合方案可显着抑制 vemurafenib 敏感和 vemurafenib 耐药黑色素瘤的细胞活力、迁移、侵袭和集落形成。维莫非尼和 b-AP15 将细胞保持在 S 期，从而导致细胞凋亡以及维莫非尼耐药 SKMEL28 细胞中自噬液泡的形成。USP14和Skp2的增强增殖作用主要是由于更有效地减少细胞凋亡和自噬。对各种蛋白质改变的进一步评估表明，与单药治疗相比，维莫非尼和 b-AP15 联合治疗中 cleaved-PARP、LC3 表达增加和 Ki67 表达减少更为明显。此外，vemurafenib和b-AP15的共同治疗可显着抑制体内耐vemurafenib黑色素瘤异种移植物的生长。总的来说，我们的研究结果表明，Skp2抑制剂和USP14抑制剂的组合为治疗BRAF抑制剂耐药性黑色素瘤提供了新的解决方案。
京公网安备 11010802027423号