Institute of Toxicology, School of Public Health, Lanzhou University, Lanzhou, 730000, China.
Cuiying Biomedical Research Center, Lanzhou University Second Hospital, Lanzhou, 730030, China.
Cell Biol Toxicol. 2023 Oct;39(5):2381-2399. doi: 10.1007/s10565-022-09729-x. Epub 2022 Jun 1.
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.
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.
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.
BRAF V600E 突变常发生于黑色素瘤中，导致肿瘤的发生。BRAF 突变驱动 RAF-MAPK-ERK 通路的过度激活。在长期使用 BRAF 抑制剂（如vemurafenib）后获得的耐药性仍然是主要障碍。先前，我们发现 E3 连接酶 Skp2 在vemurafenib 耐药的黑色素瘤细胞中过度表达，敲低 Skp2 增强了 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 在黑色素瘤治疗中具有很大的潜力；然而，其分子机制仍不清楚。
在体外，通过划痕愈合、集落形成、transwell 侵袭实验、流式细胞术、溶酶体染色和 ROS 检测研究了 vemurafenib（Vem，PLX4032）和 b-AP15 联合方案对 vem 敏感和 vem 耐药黑色素瘤的作用。在体内，使用裸鼠异种移植肿瘤模型评估了对 vem 耐药黑色素瘤的联合作用。应用 GST-pulldown 和免疫共沉淀（co-IP）实验研究了 USP14、UCHL5 和 Skp2 之间的相互作用。用环己酰亚胺（CHX）实验和泛素化实验研究了 USP14 对 Skp2 蛋白半衰期和泛素化状态的影响。
本研究揭示了 USP14 的抑制通过一种以前未被认识的机制使黑色素瘤对 vemurafenib 耐药敏感，即 USP14 而不是 UCHL5 稳定 Skp2，阻止其泛素化。Skp2 上的 K119 对于 USP14 介导的 Skp2 去泛素化和稳定是必需的。此外，USP14 上突变的催化活性氨基酸半胱氨酸（C）114 使 Skp2 的稳定作用丧失。Skp2 的稳定对于 USP14 负调控自噬是必需的。Skp2 抑制剂 vemurafenib 和 USP14/UCHL5 抑制剂 b-AP15 的联合方案显著抑制了 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。vemurafenib 和 b-AP15 将细胞阻滞在 S 期，从而导致凋亡以及在 vemurafenib 耐药 SKMEL28 细胞中形成自噬空泡。USP14 和 Skp2 增强的增殖作用主要是由于更有效地减少细胞凋亡和自噬。对各种蛋白改变的进一步评估表明，vemurafenib 和 b-AP15 联合治疗的 cleaved-PARP、LC3 的增加表达和 Ki67 的减少更为明显，比单药治疗更为明显。此外，vemurafenib 和 b-AP15 的联合治疗显著抑制了体内 vemurafenib 耐药黑色素瘤异种移植的生长。总之，我们的研究结果表明，Skp2 抑制剂和 USP14 抑制剂的联合为治疗 BRAF 抑制剂耐药黑色素瘤提供了新的解决方案。
