DAZAP1 maintains gastric cancer stemness by inducing mitophagy
Peiling Zhang, Wei Wang, Hong Xiang, Yun Zhou, Qian Peng, Guolong Liu, Zhi-Xiang Xu, Lin Lu
Peiling Zhang, Wei Wang, Hong Xiang, Yun Zhou, Qian Peng, Guolong Liu, Zhi-Xiang Xu, Lin Lu
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Research Article Cell biology Oncology Stem cells

DAZAP1 maintains gastric cancer stemness by inducing mitophagy

Abstract

Stem cells play a pivotal role in the malignant behavior of gastric cancer (GC), complicating its treatment and prognosis. However, the regulatory mechanisms of GC stem cells (GCSCs) remain poorly understood. DAZ-associated protein 1 (DAZAP1), a splicing regulator linked to various malignancies, has an unclear role in GC. This study investigated DAZAP1’s impact on GC stemness and its mechanisms. DAZAP1 promoted tumor progression in GCSCs, as shown by sphere formation assays and stemness marker analysis. Functional enrichment analysis suggested that DAZAP1 enhanced tumor stemness by promoting oxidative phosphorylation (OXPHOS), which was validated through Seahorse assays and measurements of mitochondrial potential. Transmission electron microscopy and immunofluorescence analyses demonstrated that DAZAP1 promoted mitophagy. RNA immunoprecipitation and PCR analysis revealed that DAZAP1 regulated the splicing and expression of the mitophagy-related gene ULK1 through nonsense-mediated mRNA decay. Rescue experiments showed that overexpression of ULK1 reversed the suppression of GC stemness and OXPHOS levels induced by DAZAP1 silencing. Our findings indicate that DAZAP1 reduces ULK1 decay, thereby activating mitophagy and enhancing OXPHOS to fulfill the metabolic demands of cancer stem cells. These findings highlight the therapeutic potential of DAZAP1 as a target for treating GC.

Authors

Peiling Zhang, Wei Wang, Hong Xiang, Yun Zhou, Qian Peng, Guolong Liu, Zhi-Xiang Xu, Lin Lu

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Figure 6

DAZAP1 enhances OXPHOS and maintains cell stemness in GC by inducing mitophagy.

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DAZAP1 enhances OXPHOS and maintains cell stemness in GC by inducing mit...
(A) TEM images show an increased number of mitophagosomes in DAZAP1-overexpressing (DAZAP1-OE) cells, indicating induced mitophagy. Scale bars: 1 μm (left) and 500 nm (right). (B) Confocal microscopy using MitoTracker Red reveals mitochondrial fragmentation and a reduced mitochondria-to-nucleus ratio following DAZAP1 knockdown (n = 3). (C) Immunofluorescence (IF) analysis shows increased colocalization of mitochondria with LC3B in DAZAP1-OE cells, indicating enhanced mitophagy. (D) DAZAP1 overexpression results in increased colocalization of LC3B with lysosomes, promoting autophagosome-lysosome fusion. (E) Mito-Keima plasmid labeling demonstrates increased mitophagy in DAZAP1-OE cells. Scale bars (C–E): 10 μm. (F) MitoTracker Deep Red (MTDR) dye analysis confirms impaired mitophagy in DAZAP1-knockdown cells and enhanced mitophagy in DAZAP1-OE cells. (G) Oxygen consumption rate (OCR) analysis shows reduced basal OCR, proton leak, and ATP production in GC cells treated with the autophagy inhibitor chloroquine (CQ) and the mitophagy inhibitor Mdivi-1, indicating the role of mitophagy in regulating OXPHOS. (H) Gene expression analysis reveals that Mdivi-1 restores the expression of OXPHOS complex subunit genes (UQCRC1, UQCRC2, SDHA, SDHB, ATP5F1A, ATP5F1B, and NDUFA1) elevated by DAZAP1 overexpression. (I and J) Sphere formation assay shows that Mdivi-1 treatment substantially reduces the sphere-forming ability of DAZAP1-OE cells. (K) Western blot analysis demonstrates that Mdivi-1 treatment decreases the expression of stemness markers SOX2, OCT4, and NANOG in DAZAP1-OE cells. Quantitative data are presented as the mean ± SD from at least 3 independent experiments. Statistical analysis by 1-way ANOVA followed by Tukey’s HSD post hoc test for multiple comparisons (B, G, H, and J) or unpaired Student’s t test (EV vs. OE) for comparisons between 2 groups and 1-way ANOVA followed by Tukey’s HSD post hoc test for multiple comparisons (shNC vs. sh1 and sh2) (F). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.