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.

UBL3 overexpression enhances EV-mediated Achilles protein secretion in conditioned media of MDA-MB-231 cells