线粒体自噬是自噬的选择性形式,其允许去除功能失调或过量的线粒体。这是对生理应激源的适应性反应,如缺氧,营养剥夺,或DNA损伤。线粒体自噬由特定的线粒体外膜受体促进,其中包括BNIP3和BNIP3L。线粒体自噬在癌症中的作用正在被广泛研究,更具体地说,在维持癌症干细胞(CSC)特性方面,比如自我更新。鉴于CSC是导致治疗失败和转移能力的原因,靶向线粒体自噬可能是消除CSC的一种有趣方法.在这里,我们描述了一种新的模型系统,以丰富具有高基础水平线粒体自噬的癌细胞亚群,基于BNIP3和BNIP3L的功能转录活性。简而言之,我们使用BNIP3(L)-启动子-eGFP-报告系统通过流式细胞术(FACS)分离具有高BNIP3/BNIP3L转录活性的癌细胞。该模型通过使用互补溶酶体和线粒体自噬特异性探针进行验证,以及线粒体靶向的红色荧光蛋白(RFP),即mt-Keima。高BNIP3/BNIP3L转录活性伴随着i)BNIP3/BNIP3L蛋白水平的增加,ii)溶酶体质量,和iii)基础线粒体自噬活性。此外,BNIP3/BNIP3L转录活性增加的癌细胞表现出CSC特征,如更大的乳腺球形成能力和高CD44水平。为了进一步探索模型,我们还分析了MCF7和MDA-MB-231乳腺癌细胞系的其他干性特征,直接证明BNIP3(L)-高细胞代谢活性更高,增殖性,迁徙,和抗药性,抗氧化能力提高。因此,高水平的基底线粒体自噬似乎增强了CSC的功能。
Mitophagy is a selective form of autophagy which permits the removal of dysfunctional or excess mitochondria. This occurs as an adaptative response to physiological stressors, such as hypoxia, nutrient deprivation, or DNA damage. Mitophagy is promoted by specific mitochondrial outer membrane receptors, among which are
BNIP3 and BNIP3L. The role of mitophagy in cancer is being widely studied, and more specifically in the maintenance of cancer stem cell (CSC) properties, such as self-renewal. Given that CSCs are responsible for treatment failure and metastatic capacity, targeting mitophagy could be an interesting approach for CSC elimination. Herein, we describe a new model system to enrich sub-populations of cancer cells with high basal levels of mitophagy, based on the functional transcriptional activity of
BNIP3 and BNIP3L. Briefly, we employed a
BNIP3(L)-promoter-eGFP-reporter system to isolate cancer cells with high
BNIP3/BNIP3L transcriptional activity by flow cytometry (FACS). The model was validated by using complementary lysosomal and mitophagy-specific probes, as well as the mitochondrially-targeted red fluorescent protein (RFP), namely mt-Keima. High
BNIP3/BNIP3L transcriptional activity was accompanied by increases in i)
BNIP3/BNIP3L protein levels, ii) lysosomal mass, and iii) basal mitophagy activity. Furthermore, cancer cells with increased
BNIP3/BNIP3L transcriptional activity exhibited CSC features, such as greater mammosphere-forming ability and high CD44 levels. To further explore the model, we also analysed other stemness characteristics in MCF7 and MDA-MB-231 breast cancer cell lines, directly demonstrating that BNIP3(L)-high cells were more metabolically active, proliferative, migratory, and drug-resistant, with elevated anti-oxidant capacity. Therefore, high levels of basal mitophagy appear to enhance CSC features.