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 7

DAZAP1 regulates alternative splicing of ULK1 to activate mitophagy.

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DAZAP1 regulates alternative splicing of ULK1 to activate mitophagy. (A)...
(A) PCR array results demonstrated a marked reduction in ULK1 expression in shDAZAP1 cells. (B and C) qPCR and Western blot analyses indicated that DAZAP1 overexpression upregulated ULK1 mRNA and protein levels, while knockdown downregulated them. (D) RIP-qPCR analysis confirmed DAZAP1 binding to ULK1 RNA. (E) FISH-IF staining indicated colocalization of DAZAP1 and ULK1 RNA. Scale bars: 10 μm. (F) GSEA demonstrated substantial enrichment of the spliceosome pathway in DAZAP1-OE cells. (G) RNA-seq analysis identified alternative splicing sites within ULK1 RNA, showing that DAZAP1 promotes exon 17 skipping. (H) PCR and agarose gel electrophoresis confirmed DAZAP1 regulation of ULK1 RNA splicing. (I) Actinomycin D assays demonstrated that DAZAP1 increases ULK1 mRNA stability by reducing the production of premature termination codons and subsequent nonsense-mediated decay (NMD). (J) Co-IP experiments confirmed that DAZAP1 binds to several alternative splicing factors, including HNRNPC, DDX39B, HNRNPA1L2, HNRNPM, HNRNPA1, EIF4A3, PCBP1, RBMX, and HNRNPA3. (K) Co-IP confirmed the interaction between DAZAP1 and HNRNPA1, reinforcing DAZAP1’s role in alternative splicing regulation. (L and M) IHC analysis revealed a positive correlation between DAZAP1 and ULK1 expression levels (r = 0.69, P = 1.9 × 10–5), n = 32. Scale bars: 200 μm. (N) Western blot analysis indicated that DAZAP1 overexpression upregulated mitophagy markers LC3B and P62, while knockdown downregulated them. Quantitative data are shown as the mean ± SD from a minimum of 3 independent experiments. Statistical analysis by unpaired Student’s t test (EV vs. OE) for comparisons between 2 groups and 1-way ANOVA followed by Dunnett’s multiple-comparison test (shNC vs. sh1 and sh2) (C), 1-way ANOVA followed by Dunnett’s multiple-comparison test (D), 2-way ANOVA followed by Šidák’s multiple-comparison test (I), or Spearman’s correlation analysis (M). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. NS indicates no statistically significant difference.