Department of Cellular and Molecular Anatomy, Hamamatsu University School of Medicine, 1-20-1 Handayama, Chuo-ku, Hamamatsu, Shizuoka, 431-3192, Japan.
First Department of Surgery, Hamamatsu University School of Medicine, 1-20-1 Handayama, Chuo-ku, Hamamatsu, Shizuoka, 431-3192, Japan.
Biochem Biophys Res Commun. 2024 Dec 17;738:150559. doi: 10.1016/j.bbrc.2024.150559. Epub 2024 Aug 16.
Cancer cells communicate within the tumor microenvironment (TME) through extracellular vesicles (EVs), which act as crucial messengers in intercellular communication, transporting biomolecules to facilitate cancer progression. Ubiquitin-like 3 (UBL3) facilitates protein sorting into small EVs as a post-translational modifier. However, the effect of UBL3 overexpression in EV-mediated protein secretion has not been investigated yet. This study aimed to investigate the effect of UBL3 overexpression in enhancing EV-mediated Achilles protein secretion in MDA-MB-231 (MM) cells by a dual-reporter system integrating Akaluc and Achilles tagged with Ubiquitin where self-cleaving P2A linker connects Akaluc and Achilles. MM cells stably expressing Ubiquitin-Akaluc-P2A-Achilles (Ubi-Aka/Achi) were generated. In our study, both the bioluminescence of Ubiquitin-Akaluc (Ubi-Aka) and the fluorescence of Achilles secretion were observed. The intensity of Ubi-Aka was thirty times lower, while the Achilles was four times lower than the intensity of corresponding cells. The ratio of Ubi-Aka and Achilles in conditioned media (CM) was 7.5. They were also detected within EVs using an EV uptake luciferase assay and fluorescence imaging. To investigate the effect of the UBL3 overexpression in CM, Ubi-Aka/Achi was transiently transfected into MM-UBL3-KO, MM, and MM-Flag-UBL3 cells. We found that the relative fluorescence expression of Achilles in CM of MM-UBL3-KO, MM, and MM-Flag-UBL3 cells was 30 %, 28 %, and 45 %, respectively. These findings demonstrated that UBL3 overexpression enhances EV-mediated Achilles protein secretion in CM of MM cells. Targeting UBL3 could lead to novel therapies for cancer metastasis by reducing the secretion of pro-metastatic proteins, thereby inhibiting disease progression.
癌细胞在肿瘤微环境（TME）中通过细胞外囊泡（EVs）进行通讯，EVs 作为细胞间通讯的关键信使，将生物分子运输到靶细胞中，从而促进癌症进展。泛素样 3（UBL3）作为一种翻译后修饰物，促进蛋白质分选到小 EV 中。然而，UBL3 过表达对 EV 介导的蛋白质分泌的影响尚未得到研究。本研究旨在通过整合带有泛素标签的 Akaluc 和 Achilles 的双报告系统，研究 UBL3 过表达对 MDA-MB-231（MM）细胞中 EV 介导的 Achilles 蛋白分泌的增强作用，其中自切割 P2A 接头连接 Akaluc 和 Achilles。生成了稳定表达泛素-Akaluc-P2A-Achilles（Ubi-Aka/Achi）的 MM 细胞。在我们的研究中，观察到了泛素-Akaluc（Ubi-Aka）的生物发光和 Achilles 分泌的荧光。Ubi-Aka 的强度低 30 倍，而 Achilles 的强度低 4 倍，与相应细胞的强度相比。条件培养基（CM）中 Ubi-Aka 和 Achilles 的比值为 7.5。它们也可以使用 EV 摄取荧光素酶测定法和荧光成像在 EV 中检测到。为了研究 UBL3 过表达对 CM 的影响，将 Ubi-Aka/Achi 瞬时转染到 MM-UBL3-KO、MM 和 MM-Flag-UBL3 细胞中。我们发现 MM-UBL3-KO、MM 和 MM-Flag-UBL3 细胞的 CM 中 Achilles 的相对荧光表达分别为 30%、28%和 45%。这些发现表明 UBL3 过表达增强了 MM 细胞 CM 中 EV 介导的 Achilles 蛋白分泌。通过减少促转移蛋白的分泌，靶向 UBL3 可能为癌症转移提供新的治疗方法，从而抑制疾病进展。
