ZNF454-FSTL3 axis inhibits colorectal cancer progression by inhibiting HIF-1α-mediated glycolysis in hypoxia.

BACKGROUND: Our preliminary experiments confirmed that follistatin-like 3 (FSTL3) expression is elevated in colorectal cancer (CRC) cells following CoCl(2) treatment. In this study, we investigated the regulatory role of FSTL3 in CRC progression and the molecular mechanisms underlying its high expression. METHODS: CoCl(2) (150 µM) was used to mimic hypoxia. Cell proliferation was measured by colony formation assay, wound healing assays were performed to assess cell migration, and Transwell assays were performed to evaluate invasion. The glucose metabolism pathways were assessed by detecting extracellular acidification rate, oxygen consumption rate, glucose uptake, and lactate production. The binding sites of zinc finger protein 454 (ZNF454) and FSTL3 gene promoter were analyzed by JASPAR databases, and were confirmed by chromatin immunoprecipitation (ChIP) and luciferase reporter assay. The expression levels of molecules at mRNA and protein levels were determined by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot, respectively. RESULTS: We found that both FSTL3 and hypoxia-inducible factor 1α (HIF-1α) were upregulated in CRC tissues and in CRC cells under hypoxic conditions, with a positive correlation between their expression in clinical samples. Silencing FSTL3 reduced HIF-1α expression and suppressed the expression of glycolytic enzymes (glucose transporter 1, hexokinase 2, lactate dehydrogenase A, and pyruvate kinase muscle isozyme M2). Knockdown of FSTL3 increased oxygen consumption while decreasing extracellular acidification, glucose uptake, and lactate production. Moreover, FSTL3 downregulation markedly inhibited the proliferation, migration, and invasion of CoCl(2)-treated CRC cells. We further identified ZNF454 as a potential transcription factor for FSTL3 and confirmed its binding to the FSTL3 promoter. Upregulation of ZNF454 significantly suppressed CRC cell proliferation, migration, invasion, and glycolysis, effects that were reversed by FSTL3 overexpression. In vivo, ZNF454 overexpression effectively inhibited tumor growth and reduced HIF-1α and glycolytic enzyme expression, whereas these effects were rescued by FSTL3 overexpression. CONCLUSIONS: Collectively, our findings demonstrate that ZNF454 suppresses CRC development by inhibiting FSTL3/HIF-1α-mediated glycolysis through transcriptional repression of FSTL3. This study is the first to reveal the molecular mechanism responsible for FSTL3 overexpression in CRC, providing a novel perspective for CRC treatment.