Researchers have uncovered a previously unrecognized tumor-suppressive circuit that governs stem-like behavior in hepatocellular carcinoma (HCC), the most common form of primary liver cancer. The study, led by scientists at Okayama University in Japan and published (DOI : 10.20892/j.issn.2095-3941.2025.0247) in Cancer Biology & Medicine, reveals that the protein SPRED2 binds to the tumor suppressor p53, enters the nucleus, and activates transcription of the microRNA miR-506-3p. This microRNA then directly targets KLF4 messenger RNA, reducing KLF4 protein levels and diminishing cancer stem cell (CSC) traits such as sphere formation, colony growth, invasion, and aldehyde dehydrogenase (ALDH) activity.
The findings are significant because liver cancer is the fifth most commonly diagnosed cancer and the second leading cause of cancer death worldwide. HCC accounts for 70–85% of primary liver cancers, and its five-year recurrence rate can reach 70–80%. Cancer stem cells, a small but aggressive population, drive relapse, metastasis, and chemotherapy resistance. SPRED2 is known to inhibit Ras/Raf/ERK-MAPK signaling and is downregulated in advanced cancers, including HCC. Previous work linked SPRED2 loss to higher levels of pluripotency factors such as Nanog, c-Myc, and KLF4, but how SPRED2 controls HCC stemness remained unclear.
To map the pathway, the team used HepG2, HLE, and Hep3B HCC cell lines, SPRED2-deficient and overexpressing cells, a mouse xenograft model, and 40 paired human HCC samples. They combined Western blot, RT-qPCR, co-immunoprecipitation, chromatin immunoprecipitation quantitative PCR, luciferase assays, and immunofluorescence. Clinical samples validated the cell and mouse findings. KLF4 emerged as the key pluripotency factor: knocking it down reduced sphere and colony formation, invasion, epithelial–mesenchymal transition, ALDH activity, and the percentage of CD44+ and CD90+ cells, and produced smaller tumors in mice. KLF4 knockdown also lowered Nanog and c-Myc, whereas Nanog or c-Myc knockdown did not affect KLF4, placing KLF4 upstream. SPRED2 overexpression reduced KLF4, c-Myc, and Nanog, while SPRED2 knockout increased them.
Mechanistically, miR-506-3p directly bound the 3′ untranslated region of KLF4 mRNA. In the 40 human HCC samples, KLF4 and miR-506-3p were negatively correlated, while SPRED2 and miR-506-3p were positively correlated. SPRED2 formed a nuclear complex with p53; p53 supported SPRED2 nuclear localization and bound the miR-506 promoter. SPRED2 increased miR-506-3p in a p53-dependent, dose-dependent manner. ERK and STAT3 inhibition also raised miR-506-3p, linking three pathways to KLF4 suppression. The miR-506-3p mimic reduced sphere and colony formation and ALDH activity, whereas its inhibitor increased them. In contrast, SPRED2 expression was not changed by miR-506-3p, placing SPRED2 upstream of the microRNA.
The authors said the findings reveal a previously unrecognized tumor-suppressive circuit linking SPRED2 to p53 and microRNA regulation. They said SPRED2 does not act only by blocking ERK or STAT3 signaling; it also helps p53 drive miR-506-3p, which then keeps KLF4 in check. This axis may explain why loss of SPRED2 promotes CSC-like behavior and could offer a fresh target for therapies aimed at reducing HCC stemness. Because SPRED2-driven miR-506-3p induction required functional p53, the approach may be most relevant for tumors with intact p53 signaling; HCC cells with mutant or absent p53 may need additional strategies. Future studies should test miR-506-3p mimics, SPRED2-stabilizing agents, or combination therapies in preclinical models and define which patients are most likely to benefit. Such work could help translate this newly defined axis into precision approaches for HCC.
The research was supported in part by the Japan Society for the Promotion of Science (Grant Nos. 25293095, 90264283, and 22K19562). The full study is available in Cancer Biology & Medicine, an open-access journal indexed in SCOPUS, MEDLINE, and SCI, with full texts freely visible to clinicians and researchers worldwide via http://www.ncbi.nlm.nih.gov/pmc/journals/2000/.
