SPRED2 loss drives liver cancer stemness via p53/miR-506-3p/KLF4
image: Schematic diagram indicating the mechanisms by which SPRED2 regulates the stemness of HCC cells. miR-506-3p downregulates KLF4 expression. Left panel: Under normal conditions, SPRED2 binds to RAF and/or RSK2 and inhibits the ERK signaling pathway. SPRED2 also binds to DYRK1A and inhibits the STAT3 signaling pathway. As a result, the inhibitory effect on miR-506-3p is negated, resulting in increased expression of miR-506. SPRED2 also forms a protein complex with p53 and directly promotes miR-506-3p expression. Thus, SPRED2 coordinates all three pathways (ERK, STAT3, and p53) to increase miR-506-3p expression, thereby reducing KLF4 expression and cell stemness in HCC cells. Right panel: When HCC cells lose the expression of SPRED2, the ERK and STAT3 pathways are no longer inhibited and the formation of SPRED2/p53 protein complex decreases, leading to decreased miR-506-3p expression. This results in increased KLF4 expression and promotion of HCC stemness.
Credit: Cancer Biology & Medicine
Researchers have identified a new tumor-suppressive circuit that controls stem-like behavior in hepatocellular carcinoma (HCC). The work shows that Sprouty-related, EVH1 domain-containing protein 2 (SPRED2) binds the tumor suppressor p53, enters the nucleus, and activates transcription of the microRNA miR-506-3p. In turn, miR-506-3p directly targets KLF4 messenger RNA, lowering KLF4 protein and reducing cancer stem cell (CSC) traits, including sphere formation, colony growth, invasion, and aldehyde dehydrogenase (ALDH) activity. The study defines a SPRED2/p53/miR-506-3p/KLF4 axis and suggests that restoring this pathway could help suppress HCC progression, recurrence, and treatment resistance.
Liver cancer is the fifth most commonly diagnosed cancer and the second leading cause of cancer death worldwide. Hepatocellular carcinoma (HCC) accounts for 70–85% of primary liver cancers, and its five-year recurrence rate can reach 70–80%. Cancer stem cells (CSCs), a small but aggressive cell population, drive relapse, metastasis, and chemotherapy resistance. SPRED2 inhibits Ras/Raf/extracellular signal-regulated kinase–mitogen-activated protein kinase (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. Given these challenges, or because of these issues, in-depth research is needed on the molecular mechanisms by which SPRED2 regulates HCC stemness.
The study was led by researchers at Okayama University in Japan and published (DOI : 10.20892/j.issn.2095-3941.2025.0247) in Cancer Biology & Medicine. Using HepG2, HLE, and Hep3B HCC cell lines, SPRED2-deficient and overexpressing cells, a mouse xenograft model, and 40 paired human HCC samples, the team combined Western blot, reverse transcription quantitative polymerase chain reaction (RT-qPCR), co-immunoprecipitation (Co-IP), chromatin immunoprecipitation quantitative polymerase chain reaction (ChIP-qPCR), luciferase assays, and immunofluorescence to map the pathway. Clinical samples validated the cell and mouse findings.
KLF4 emerged as the key pluripotency factor. Knocking down KLF4 in HepG2 cells reduced sphere and colony formation, invasion, epithelial–mesenchymal transition (EMT), aldehyde dehydrogenase (ALDH) activity, and the percentage of cluster of differentiation 44–positive (CD44+) and cluster of differentiation 90–positive (CD90+) cells; it also produced smaller tumors in mice. KLF4 knockdown 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 (3′-UTR) of KLF4 mRNA. In 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 signal transducer and activator of transcription 3 (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. These results show SPRED2 coordinates ERK, STAT3, and p53 signaling to reduce HCC stemness.
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. They said 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. They said the pathway may be especially important because it connects three signaling routes to a single stemness regulator.
Targeting the SPRED2/p53/miR-506-3p/KLF4 axis may provide new therapeutic strategies for HCC. Restoring miR-506-3p or SPRED2 activity could lower KLF4 and inhibit CSC traits, potentially reducing relapse, metastasis, and chemoresistance. 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.
###
References
DOI
10.20892/j.issn.2095-3941.2025.0247
Original Source URL
https://doi.org/10.20892/j.issn.2095-3941.2025.0247
Funding information
This work was supported in part by Japan Society for the Promotion of Science (Grant Nos. 25293095, 90264283, and 22K19562).
About Cancer Biology & Medicine
Cancer Biology & Medicine (CBM) is a peer-reviewed open-access journal sponsored by China Anti-cancer Association (CACA) and Tianjin Medical University Cancer Institute & Hospital. The journal monthly provides innovative and significant information on biological basis of cancer, cancer microenvironment, translational cancer research, and all aspects of clinical cancer research. The journal also publishes significant perspectives on indigenous cancer types in China. The journal is indexed in SCOPUS, MEDLINE and SCI (IF 12.4), with all full texts freely visible to clinicians and researchers all over the world (http://www.ncbi.nlm.nih.gov/pmc/journals/2000/).