Wang Xuan, Qi Guocui, Yang Kunning, Ma Linlin, Kang Yuansai, Tang Xiaojing, Han YanTao
Department of Pharmacology, School of Basic Medicine, Qingdao University, 308 Ningxia Road, Qingdao, 266071, China.
Department of Respiratory Medicine, Weifang Second People's Hospital, Weifang, China.
J Transl Med. 2025 May 14;23(1):544. doi: 10.1186/s12967-025-06530-2.
Triple-negative breast cancer (TNBC) is a subtype with the worst prognosis and there is still a lack of effective treatment. Exosomes (Exos) secreted by cancer cells to tumor microenvironment play an important role in cancer progression. We have demonstrated that the function of Rasal2 in the modulation of breast cancer progression is exos-mediated, but the relationship between Rasal2 and exosome secretion remains elusive.
Rasal2 knock-out (KO) MDA-MB-231 cells were conducted by crispr-cas9 technique and Rab27a knock-down (KD) in Rasal2 KO MDA-MB-231 cells (KO + KD) were further established by siRNA-mate plus transfection Reagent. Control (CT)/KO MDA-MB-231 cells stably overexpressing GFP-LC3 were generated by using GFP-LC3 plamisd. Transmission electron microscope (TEM), nanoparticle tracking analysis (NTA) and western blot analysis (WB) were used to identify exos derived from TNBC. Confocal microscopy was used to observe the autophagic flux and the colocalization of autophagosomes and multivesicular bodies (MVBs). Co-immunoprecipitation analysis was performed to determine the interaction between Rasal2 and Rab27a. Immunohistochemical analysis were used to detect the expression levels of autophagy-related proteins in tumor tissues of xenograft mice inoculated with CT/KO/KO + KD MDA-MB-231 cells.
In this paper, we found that Rasal2 KO disrupts autophagic flux and induces secretory autophagy to promote autophagic-exos secretion in TNBC. Moreover, Rasal2 inhibits the activity of Rab27a which regulates vesicles transport and fusion, and Rab27a mediates Rasal2 KO-induced autophagic-exos secretion. Additionally, Rab27a KD inhibits Rasal2 KO-induced secretory autophagy, thereby promoting TNBC progression both in vivo and in vitro.
Collectively, these findings delineated the role of Rab27a in TNBC progression modulated by Rasal2 through autophagy-exos pathway and suggested that it is of great significance for the early diagnosis, targeted therapy and prognosis judgement of TNBC from the perspective of tumor microenvironment.
三阴性乳腺癌（TNBC）是预后最差的一种亚型，目前仍缺乏有效的治疗方法。癌细胞分泌到肿瘤微环境中的外泌体（Exos）在癌症进展中起重要作用。我们已经证明Rasal2在调节乳腺癌进展中的功能是由外泌体介导的，但Rasal2与外泌体分泌之间的关系仍不清楚。
采用crispr-cas9技术构建Rasal2基因敲除（KO）的MDA-MB-231细胞，并通过siRNA-mate加转染试剂在Rasal2基因敲除的MDA-MB-231细胞（KO + KD）中进一步敲低Rab27a。使用GFP-LC3质粒构建稳定过表达GFP-LC3的对照（CT）/KO MDA-MB-231细胞。通过透射电子显微镜（TEM）、纳米颗粒跟踪分析（NTA）和蛋白质免疫印迹分析（WB）鉴定源自TNBC的外泌体。利用共聚焦显微镜观察自噬流以及自噬体与多囊泡体（MVBs）的共定位。进行免疫共沉淀分析以确定Rasal2与Rab27a之间的相互作用。采用免疫组织化学分析检测接种CT/KO/KO + KD MDA-MB-231细胞的异种移植小鼠肿瘤组织中自噬相关蛋白的表达水平。
在本文中，我们发现Rasal2基因敲除会破坏自噬流并诱导分泌性自噬，从而促进TNBC中自噬外泌体的分泌。此外，Rasal2抑制调节囊泡运输和融合的Rab27a的活性，并且Rab27a介导Rasal2基因敲除诱导的自噬外泌体分泌。此外，Rab27a基因敲低抑制Rasal2基因敲除诱导的分泌性自噬，从而在体内和体外促进TNBC进展。
总的来说，这些发现阐明了Rab27a在Rasal2通过自噬外泌体途径调节TNBC进展中的作用，并表明从肿瘤微环境的角度来看，这对TNBC的早期诊断、靶向治疗和预后判断具有重要意义。
