Research ArticleExperimental Studies
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PPIL1 Drives Hepatocellular Carcinoma Progression and Cancer Stem Cell Self-renewal Through DAAM2-mediated Wnt/β-Catenin Activation

JIAYI WU, SHIYUAN CHANG, SHUO CHEN, YU QI and WEIJUN SU
Cancer Genomics & Proteomics September 2025, 22 (5) 760-774; DOI: https://doi.org/10.21873/cgp.20535

Abstract

Background/Aim: Hepatocellular carcinoma (HCC) accounts for ~90% of primary liver cancer, which ranks as the third-leading cause of global cancer mortality. Emerging evidence establishes cancer stem cells (CSCs) as central regulators of HCC progression, metastasis, and therapeutic resistance, with stemness-related pathways like Wnt/β-catenin signaling critically maintaining CSC self-renewal. In this study, we aimed to investigate the role of Peptidyl-prolyl isomerase-like 1 (PPIL1) in HCC progression and CSC self-renewal.

Materials and methods: PPIL1 expression patterns were systematically analyzed using The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA-LIHC) data and validated in primary HCC specimens via qRT-PCR and western blot. PPIL1 was knocked down in HCC cell lines using shRNAs, and cell viability, migration, and sphere formation were assessed in vitro. Xenograft mouse models were established to evaluate the effects of PPIL1 on tumor growth kinetics and liver CSC-related properties. Transcriptome analysis was performed to identify downstream targets and signaling pathways affected by PPIL1 knockdown.

Results: Our analysis revealed significantly elevated PPIL1 expression in HCC tumors and liver CSCs, with its expression level positively correlating with tumor stage and histological grade. PPIL1 knockdown effectively suppressed HCC cell proliferation, migration, and in vivo tumor growth. The essential role of PPIL1 in liver CSC maintenance was demonstrated by impaired sphere-forming capacity and diminished tumor initiation potential. Mechanistic studies identified PPIL1 as a regulator of Wnt/β-catenin signaling through transcriptional up-regulation of dishevelled associated activator of morphogenesis 2 (DAAM2).

Conclusion: Our findings suggest PPIL1 to be a crucial regulator of HCC progression and liver CSC maintenance via DAAM2-mediated Wnt/β-catenin activation. This positions PPIL1 as a promising molecular target for HCC therapy, with particular relevance for addressing CSC-driven therapeutic resistance.

Keywords:

Introduction

Liver cancer remains the third leading cause of cancer-related mortality worldwide, representing an unresolved global health burden (1). Hepatocellular carcinoma (HCC), which constitutes approximately 90% of primary liver cancers, is characterized by aggressive progression, limited therapeutic efficacy, and frequent recurrence (2-4). Despite advancements in multimodal treatments (e.g., surgical resection, tyrosine kinase inhibitors, and immune checkpoint blockade), the five-year survival rate for advanced HCC remains below 20% (5, 6), with over 70% of patients developing resistance to systemic therapies within one year (7, 8). This therapeutic stagnation underscores the critical need to unravel the molecular mechanisms driving HCC relapse and chemoresistance, which are largely attributed to tumor heterogeneity (9, 10).

Recent studies have highlighted the pivotal role of cancer stem cells (CSCs), a self-renewing malignant subpopulation with intrinsic plasticity and therapy resistance, in sustaining tumor heterogeneity and driving cancer progression, including HCC (11-16). Within HCC, CSCs are predominantly identified through functional assays (e.g., sphere formation) that reliably assess stem-like properties, complemented by surface markers such as CD133, CD13, EpCAM, CD24, CD90, and CD44 (17-19). CSCs are characterized by the constitutive activation of phylogenetically conserved stemness pathways, including Wnt/β-catenin, Notch, Hedgehog, and Hippo/Yap, which coordinately regulate their survival, self-renewal, and tumorigenic competence (20-23). In HCC and other malignancies, CSCs exhibit marked resistance to both conventional (e.g., radiotherapy, chemotherapy) and emerging therapies (e.g., CAR-T, immune checkpoint inhibitors) (24-27). Despite their established role as key drivers of therapeutic resistance and tumor recurrence in HCC, the specific regulatory network governing CSC maintenance and plasticity remains poorly characterized, limiting the development of CSC-targeted therapies for this malignancy.

Peptidyl-prolyl isomerase-like 1 (PPIL1), a member of the PPIase family, functions as a spliceosome component that facilitates pre-mRNA splicing. Emerging evidence suggests that PPIL1 may exert oncogenic functions, as its expression is up-regulated in colorectal cancer cells, and siRNA-mediated PPIL1 knockdown (KD) significantly reduces tumor cell viability (28). Furthermore, Janneh et al. revealed that PPIL1 acts as a signaling hub in tumor metastasis by forming a S1PR1/C3/PPIL1/NLRP3 axis, highlighting the dual roles of PPIL1 in both tumor proliferation and dissemination (29). According to the Human Protein Atlas (https://www.proteinatlas.org/ENSG00000137168-PPIL1), PPIL1 has been identified as an unfavorable prognostic marker in liver cancer (30). The survival analysis demonstrates a correlation between increased PPIL1 expression and poor prognosis in liver cancer patients. Despite these advances, direct mechanistic insights into PPIL1 in HCC are lacking. Intriguingly, multiple studies demonstrate that other PPIases regulate HCC progression through multifaceted mechanisms. For instance, Cyclophilin A (CypA) acts as a critical host factor for Hepatitis C Virus (HCV) replication and promotes HCC metastasis by up-regulating MMP3 and MMP9 (31-33). Notably, CypA inhibitors have been developed to target HCV-induced HCC, with their anticancer effects partially independent of antiviral activity (34, 35). Moreover, peptidylprolyl cis/trans isomerase, NIMA-interacting 1 (PIN1), is overexpressed in HCC and plays a pivotal role in hepatocarcinogenesis via the up-regulation of cyclin D1, as well as the interactions with the x-protein of hepatitis B virus (HBx) and exportin 5 (36-38). These findings collectively suggest a conserved oncogenic role for the PPIase family that may extend to PPIL1 in HCC pathogenesis. Thus, in this study, we evaluated the expression of PPIL1 in human HCC and its role in HCC hepatocarcinogenesis and self-renewal of liver CSCs via activating the DAAM2-Wnt signaling axis.

Materials and Methods

Antibodies and reagents. Anti-PPIL1 (cat. no. 15144-1-AP) antibody was obtained from Proteintech Group (Wuhan, PR China). Anti-β-actin (cat. no. 4970) antibody was purchased from Cell Signaling Technology (Danvers, MA, USA). Phycoerythrin (PE)-conjugated CD133 (cat. no. 130098826) antibody was from Miltenyi Biotec (Bergisch Gladbach, Germany). Fluorescein isothiocyanate (FITC)-conjugated CD13 antibody (cat. no. 11-0138) was from eBioscience (San Diego, CA, USA). N2 supplement and B27 supplement were from Invitrogen (Carlsbad, CA, USA). Ultra-low attachment plates (cat. no. 3471) were from Corning Company (Corning, NY, USA).

Human samples and cells. This work was approved by the ethics committee of Nankai University (NKUIRB2025066). HCC tissues used in this work were obtained from The First Affiliated Hospital of Zhengzhou University. HEK293T, Huh7, and Hep3B cells were obtained from Pingping Zhu’s lab (Zhengzhou University). Cells were cultured in high-glucose Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 100 μg/ml streptomycin, 100 U/ml penicillin, and 10% fetal bovine serum (FBS). All cell lines were maintained at 37°C in a humidified atmosphere containing 5% CO2 under standard static culture conditions. For the preparation of HCC primary cells, HCC samples were immediately obtained after resection. Tumor bulk was cut into 1 mm3 pieces with scissors, followed by digestion with collagenase IV. The samples were then passed through a 70 μm cell strainer and centrifuged at 50 g at 4°C for 10 min. The supernatant was collected and further centrifuged at 300 g at 4°C for 10 min. HCC cells were enriched in the pellets, followed by red blood cell elimination.

Animals. This work was approved by the ethics committee of Nankai University. For all mouse experiments, 6-week-old male BALB/c nude mice were purchased from Henan Skobes Biotechnology Co., Ltd (Anyang, PR China). Mice were maintained under SPF conditions, housed in groups of 5-6 per cage with a 12-hour light/dark cycle, and provided with controlled temperature and humidity. All mice were randomly grouped, and no mice were excluded from analyses. No tumor burden was over 1800 mm3. All efforts to minimize animal suffering were made.

Lentivirus packaging and the generation of stable cell lines. For the generation of PPIL1 knockdown stable cell lines, sequences of shRNAs targeting PPIL1 were synthesized and cloned into the pSiCoR vector (cat no. 12084, Addgene). shRNA sequences for PPIL1 used in this study are listed in Supplementary Table I. For lentivirus packaging, we transfected HEK293T cells with pSiCoR and packaging plasmids (4 μg pSiCoR vector, 1 μg VSVG, 1 μg RRE, and 2 μg RSV-REV for each 10 cm dish). Huh7 and Hep3B cells were infected with lentivirus supernatants. Briefly, target cells were seeded into 6-well plates at a density of 105/well one night prior to infection. On the day of infection, 1 ml filtered lentiviral supernatant was mixed with 2 ml fresh complete medium containing 8 μg/ml polybrene, added to the target cells, and centrifuged at 2,000 rpm for 30 min at room temperature. Following the centrifuge, cells were incubated overnight, and the medium was replaced 24 h post-infection. After screening with puromycin at 8 μg/ml from 48-h post-infection for 5 days, stable cell lines were established. Both qRT-PCR analysis and western blot were applied to validate the knockdown efficiency.

For PPIL1 overexpression stable cell line generation, the human PPIL1 cDNA was cloned into pLVX-IRES-ZsGreen1 vector containing a ZsGreen1 reporter. Lentivirus packaging was performed similarly as mentioned above. Huh7 and Hep3B cells were infected with viral supernatants similarly as mentioned above, and GFP-positive cells were monitored by fluorescence microscopy. Since GFP fluorescence was observed in approximately all cells by 48-h post-infection, FACS sorting was omitted. Overexpression efficiency was further confirmed by qRT-PCR.

RNA extraction and qRT-PCR analysis. Total RNA samples were isolated using TRIzol. 1 μg of RNA was reverse transcribed into cDNA and then subjected to quantitative real-time PCR analysis with ABI QuantStudio 5 (Thermo Fisher Scientific, Waltham, MA, USA). Relative changes in expression levels were calculated. qRT-qPCR primers are listed in Supplementary Table II.

Western blot. Protein samples were loaded onto a 12% SDS-polyacrylamide gel and separated by electrophoresis, then transferred onto a PVDF membrane. After blocking, the membranes were incubated with primary antibodies at 4°C overnight. After washing, the membranes were incubated with appropriate secondary antibodies at room temperature for one hour. Finally, the membranes were washed, and the protein bands were visualized using ECL reagents.

Cell viability assay. Cell viability was assessed using the CCK-8 kit according to the manufacturer’s instructions. Hepatocellular carcinoma cells were cultured in 96-well plates at a density of 2,000 cells per well. 10 μl of CCK-8 reagent (Selleck Chemicals, Houston, TX, USA) was added and incubated for one hour. Absorbance was measured at 450 nm.

Wound healing assay. Cells were plated in six-well plates. Then, the cells were gently scraped using a fresh pipette tip, rinsed multiple times with phosphate-buffered saline (PBS), and incubated in serum-free DMEM for 24 hours. The migrated area of the cells was photographed under a microscope and analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Sphere formation assay. 2,000 Huh7 cells were grown in sphere formation medium (DMEM supplemented with 20 ng/ml bFGF, 20 ng/ml EGF, N2 and B27). Two weeks later, spheres were counted, and photographs were taken. For primary cells, 5,000 cells were cultured per well, and other procedures were consistent with those for Huh7 cells.

Xenograft growth in nude mice. In subcutaneous injection models, 1×107 control, PPIL1 knockdown or PPIL1 overexpressing cells were implanted bilaterally into the posterior dorsal flank regions of 6-week-old male BALB/c nude mice. Tumors were measured every four days starting from day 10. After 26 days, the mice were sacrificed, the tumors were isolated, and photographs were taken.

Diluted xenograft tumor formation. 10, 1×102, 1×103, 1×104 or 1×105 control or PPIL1 knockdown cells were injected into the posterior dorsal flank regions of 6-week-old male BALB/c nude mice. Tumor formation was evaluated three months post-implantation, followed by the calculation of ratios of tumor-free mice.

Statistical analysis. Differential expression analysis and Kaplan-Meier curves were generated by GEPIA 2 (Gene Expression Profiling Interactive Analysis) and UALCAN based on the TCGA-LIHC cohort (39-41).

Data were analyzed using unpaired t-tests (two-group comparisons) or ANOVA (multi-group comparisons), followed by appropriate post hoc tests when significant differences were detected. Statistical significance was set at p<0.05.

Results

PPIL1 is highly expressed in HCC. To elucidate the clinical significance of PPIL1 in HCC, we first analyzed its expression profile using TCGA-LIHC cohort data (n=374). The analysis revealed that high PPIL1 expression was significantly correlated with poorer overall survival (OS) and shorter disease-free survival (DFS) compared to low-expression groups (Figure 1A, B). Furthermore, integrated transcriptomic analysis demonstrated marked up-regulation of PPIL1 in tumor tissues relative to adjacent normal tissues, with expression levels showing a positive correlation with advancing tumor stage and histological grade (Figure 1C-E).

Figure 1.
Figure 1.

PPIL1 is highly expressed in HCC. (A and B) PPIL1 is correlated with the poor prognosis of HCC. (A) Overall survival (OS) and (B) disease-free survival (DFS) analyses of HCC patients from the TCGA-LIHC cohort, stratified based on the median expression level of PPIL1 (high vs. low, n=182 per group). (C) Elevated PPIL1 expression in HCC tumor tissues from the TCGA-LIHC cohort. (D-E) PPIL1 expression levels in HCC tissues stratified by (D) clinical stage and (E) histological grade versus normal liver controls (TCGA-LIHC). (F-G) Experimental validation of PPIL1 overexpression in HCC. (F) qRT-PCR and (G) western blot analysis of paired primary samples (P: peri-tumor, T: tumor). β-actin served as loading control. For data in (F), n=3 independent experiments. Data are shown as mean±standard deviation (SD); *p<0.05, **p<0.01 and ***p<0.001 using t-test.

Based on these TCGA findings, we proceeded to validate PPIL1 expression patterns in primary HCC specimens through qRT-PCR and western blot analyses. The experimental results consistently confirmed significant overexpression of PPIL1 at both mRNA and protein levels in HCC tumor tissues compared to peri-tumor tissues (Figure 1F, G).

PPIL1 knockdown suppresses HCC cell proliferation and migration. To investigate the functional consequences of PPIL1 depletion in hepatocellular carcinoma, we performed loss-of-function studies both in vitro and in vivo. Efficient knockdown of PPIL1 in Huh7 and Hep3B cell lines was first confirmed at both transcriptional (Figure 2A and Supplementary Figure 1A) and translational levels (Figure 2B and Supplementary Figure 1B). Subsequent CCK-8 assays demonstrated that PPIL1 knockdown significantly impaired the proliferative capacity of both HCC cell lines (Figure 2C and Supplementary Figure 1C). Wound healing assays further revealed that PPIL1 knockdown markedly attenuated the migratory potential of HCC cells (Figure 2D). To extend these findings to an in vivo setting, we established xenograft models by subcutaneously injecting PPIL1-knockdown or control shRNA-transfected Hep3B cells into BALB/c nude mice. Longitudinal monitoring demonstrated that PPIL1 knockdown led to significant suppression of tumor growth compared to the control group (Figure 2E). Collectively, these results establish PPIL1 as a critical regulator of HCC cell proliferation and migration.

Figure 2.
Figure 2.

PPIL1 knockdown suppresses HCC proliferation and migration. (A-B) Establishment of PPIL1-knockdown Huh7 cells using pSiCoR lentivirus, validated by (A) qRT-PCR and (B) western blot. (C) CCK-8 assay showing the viability of PPIL1-knockdown cells. (D) Representative images (left panel) and the quantification (right panel) of wound healing assay demonstrating migration capacity (left panel: scale bars=500 μm). (E) In vivo tumor growth curves and tumor images after subcutaneous injection of 1×107 PPIL1-knockdown/control Hep3B cells into BALB/c nude mice. Data are shown as mean±standard deviation (SD); *p<0.05, **p<0.01 and ***p<0.001 using t-test and repeated measures ANOVA followed by the Bonferroni post hoc test.

PPIL1 sustains liver CSC self-renewal and tumor-initiating capacity. Liver CSCs exhibit intrinsic tumor-initiating capacity and resistance to conventional therapies. Their stemness maintenance mechanisms, however, remain poorly understood. In previous work, we validated that CD13+CD133+ HCC cells exhibit characteristics of liver CSCs, whereas CD13−CD133− cells represent non-CSC populations (22). Using fluorescence-activated cell sorting (FACS) of clinically obtained primary HCC specimens, we identified consistent PPIL1 up-regulation in CD13+CD133+ CSCs compared to CD13−CD133− non-CSCs (Figure 3A). This expression pattern was recapitulated in patient-derived oncosphere cultures (Figure 3B), suggesting the intrinsic association of PPIL1 with the liver CSC population.

Figure 3.
Figure 3.

PPIL1 sustains liver CSC self-renewal and tumor-initiating capacity. (A and B) Elevated expression of PPIL1 in liver CSCs. qRT-PCR and western blot were performed to compare the expression levels of PPIL1 in liver CSCs (CD13+CD133+) and non-CSCs (CD13−CD133−) (A), or in oncospheres and non-spheres (B). Left: n=3 independent experiments. Right: β-actin served as loading control. (C) Typical sphere images and (D) sphere-formation ratios of PPIL1 knockdown and control Huh7 cells. For images in (C), scale bars=200 μm. For (D), n=3 cell cultures. (E) Sphere-formation ratios of serial sphere formation. n=3 cell cultures. (F) Ratios of tumor-free mice after 3 months of tumor formation after injection of increasing numbers of PPIL1 knockdown and control cells. n=6 mice for each group. Data are shown as mean±standard deviation (SD); *p<0.05, **p<0.01 and ***p<0.001 using t-test and repeated measures ANOVA followed by Dunnett’s post hoc test.

Building upon the identified association between PPIL1 and liver CSCs, we next performed functional assays to investigate the role of PPIL1 in liver CSC maintenance. In vitro sphere formation assays demonstrated that PPIL1 knockdown significantly compromised primary and serial sphere-forming capacity (Figure 3C-E), indicative of impaired CSC self-renewal potential. Moreover, limiting dilution xenograft analysis provided definitive evidence that PPIL1 knockdown reduced tumor-initiating cell frequency in vivo (Figure 3F), confirming its indispensable role in tumor initiation. Collectively, these findings demonstrate that PPIL1 is required for both the self-renewal and tumor-initiation capacities of liver CSCs.

Oncogenic effects of PPIL1 overexpression through enhanced proliferation, migration, and stemness properties. To further validate the functional significance of PPIL1 in hepatocellular carcinoma, we generated PPIL1-overexpressing HCC cell lines (Figure 4A). Notably, PPIL1 overexpression not only accelerated cell proliferation in both monolayer culture (Figure 4B) and xenograft models (Figure 4C), but also markedly enhanced migratory capacity, as evidenced by wound healing assays (Figure 4D).

Figure 4.
Figure 4.

Oncogenic effects of PPIL1 overexpression through enhanced proliferation, migration, and stemness properties. (A) PPIL1 overexpressed Huh7 cells were established and confirmed by qRT-PCR. n=3 independent experiments. (B) CCK-8 assay showing the viability of PPIL1-overexpressing (oePPIL1) cells. n=3 independent experiments. (C) In vivo tumor growth curves after subcutaneous injection of 1×107 PPIL1-knockdown/control Hep3B cells into BALB/c nude mice. n=5 mice for each group. (D) Representative images (left panel) and the quantification (right panel) of wound healing assay demonstrating migration capacity. Scale bars=500 μm. n=3 independent experiments. (E) Typical sphere images and (F) sphere-formation ratios of PPIL1–overexpressing cells. For images in (E), scale bars=200 μm. For (F), n=3 cell cultures. (G) Sphere-formation ratios of serial sphere formation. n=3 cell cultures. Data are shown as mean±standard deviation (SD), *p<0.05 and **p<0.01 using t-test and repeated measures ANOVA followed by the Bonferroni post hoc test.

More importantly, PPIL1 overexpression profoundly augmented CSC-like properties. Primary and serial sphere formation assays revealed that PPIL1-overexpressing cells exhibited significantly increased oncosphere-forming efficiency (Figure 4E, F) and sustained self-renewal capability (Figure 4G), suggesting its pivotal role in maintaining the stem cell compartment. These gain-of-function studies, combined with our previous loss-of-function data, collectively demonstrate PPIL1 to be a critical regulator of HCC cell stemness and tumorigenesis.

PPIL1 activates the Wnt signaling pathway by up-regulating DAAM2. To investigate the molecular mechanism by which PPIL1 regulates the stemness of HCC cells, we performed transcriptome profiling of PPIL1-knockdown HCC cells (GEO accession number: GSE296307). Comparative analysis identified 553 differentially expressed genes (DEGs) with |log2FC| >1 and adjusted p<0.05 (303 up-regulated and 250 down-regulated), as illustrated by the volcano plot in Figure 5A. KEGG pathway enrichment analysis revealed the Wnt signaling pathway as the most significantly altered pathway (p=0.002) (Figure 5B).

Figure 5.
Figure 5.

PPIL1 activates the Wnt signaling pathway by up-regulating DAAM2. (A) Volcano plot of RNA-seq data showing the distribution of mapped differentially expressed genes between the control and PPIL1 knockdown Huh7 cells. (B) KEGG pathway enrichment analysis of RNA-seq data from PPIL1 knockdown versus control Huh7 cells showing significantly enriched pathways. (C) Expression levels of DAAM2 and other main self-renewal pathway target genes in PPIL1 knockdown cells detected by qRT-PCR. n=3 independent experiments. Data are shown as means±standard deviation (SD). *p<0.05, **p<0.01, ***p<0.001 and p>0.05 for non-significant (ns), using repeated measures ANOVA followed by Dunnett’s post hoc test.

Further investigation identified DAAM2 (dishevelled associated activator of morphogenesis 2), a known modulator of canonical Wnt signaling during embryonic development (42), as a key downstream effector of PPIL1. qRT-PCR validation confirmed significant down-regulation of DAAM2 expression in PPIL1-knockdown cells (Figure 5C). To confirm that PPIL1 specifically regulates the Wnt signaling pathway rather than other stemness-related pathways, we further examined downstream targets of multiple pathways and observed significant down-regulation of Wnt target genes (MYC, CCND1), while no notable changes were detected in Hedgehog, Notch, or Hippo/YAP signaling components (Figure 5C). These results collectively demonstrate that PPIL1 maintains liver CSC properties through transcriptional up-regulation of DAAM2. This newly identified PPIL1-DAAM2-Wnt signaling axis provides mechanistic insight into the regulation of cancer stemness in HCC.

Discussion

To our knowledge, this study provides the first experimental evidence implicating PPIL1 in hepatocarcinogenesis. We demonstrate that PPIL1 is overexpressed in HCC and correlates with advanced disease stage and poor patient outcomes. Functional analyses revealed that PPIL1 depletion suppresses tumor cell proliferation, migration, and cancer stem cell self-renewal, while its overexpression exacerbates malignant phenotypes. Mechanistically, PPIL1 appears to regulate HCC progression through DAAM2-mediated Wnt pathway activation. Although these preliminary findings require further validation, our work establishes PPIL1 as a previously unrecognized player in HCC biology and highlights its potential as a therapeutic target to circumvent CSC-mediated therapy resistance, which is a hallmark of treatment failure in HCC.

Cancer stem cells have been verified in a variety of solid tumors (43, 44). CSCs share fundamental parallels with normal stem cells, particularly in their capacity for self-renewal (45, 46). These CSCs within tumor bulk are called the seeds of tumorigenesis and display the capacity to initiate a new tumor (47). The stemness phenotype in CSCs is driven by dysregulated core developmental pathways including Wnt/β-catenin (20), Notch (21), Hedgehog (22), and Hippo/Yap (23) signaling, which exhibit persistent activation through oncogenic rewiring of transcriptional networks.

Aberrant activation of the Wnt/β-catenin pathway drives hepatocellular carcinoma initiation and progression while sustaining liver CSC self-renewal, metastatic potential, and intrinsic therapy resistance (48-53). In the past decade, epigenetic factors and non-coding RNAs have also been reported to modulate the expression of Wnt/β-catenin components, transcription factors (TCF), APC, and β-catenin, resulting in CSC self-renewal (54). In this study, we identified PPIL1 as a novel upstream regulator of the Wnt pathway, where it transcriptionally up-regulates DAAM2, a dual-functional effector implicated in both canonical and non-canonical Wnt signaling, to enforce liver CSC stemness. While DAAM2 serves as a context-dependent signaling hub that bridges Wnt pathway branches through mechanisms such as phosphorylation-dependent conformational changes, its role in HCC remains poorly characterized (55, 56). The precise mechanisms underlying PPIL1-mediated transcriptional regulation of DAAM2 remain to be elucidated. Analogous to other cyclophilin family members (e.g., CypA), PPIL1 may interact with specific transcription factors to modulate DAAM2 transcription, possibly by affecting the structural conformation or activity of transcriptional complexes (57, 58). Furthermore, as a potential RNA-binding protein, PPIL1 might regulate DAAM2 transcription through RNA-centric mechanisms, such as stabilizing DAAM2-associated circular RNAs (circRNAs) or interacting with non-coding RNAs to recruit epigenetic modifiers that enhance DAAM2 promoter activity.

Therapeutic resistance and recurrence after tumor treatment are universal in chemotherapy, radiotherapy, targeted therapy, and immunotherapy. CSCs are capable of escaping cell death under such therapeutic stress (59). Lenvatinib, an inhibitor of multiple kinases, serves as a typical drug for HCC. Liver CSCs activate Wnt/β-catenin and Hippo signaling pathways to harbor lenvatinib resistance (60, 61). In this study, we identified PPIL1 as a promoter of hepatocarcinogenesis and liver CSC self-renewal via activating Wnt signaling. The combination of lenvatinib with PPIL1 inhibition may effectively suppress CSC-mediated drug resistance and reduce tumor recurrence in hepatocellular carcinoma therapy.

Conclusion

This study establishes PPIL1 as a crucial regulator of hepatocellular carcinoma progression and liver CSC maintenance. Mechanistically, PPIL1 activates the Wnt/β-catenin signaling pathway through transcriptional up-regulation of DAAM2, thereby promoting HCC cell proliferation, migration, and CSC self-renewal capacity. PPIL1 emerges from these findings as a viable molecular target in HCC therapy, showing particular promise for addressing therapeutic resistance driven by cancer stem cells.

Acknowledgements

We thank the staff at Department of Thoracic Surgery, the First Affiliated Hospital of Zhengzhou University for assisting in sample collection.

Footnotes

  • Supplementary Material

    Supplementary figures and tables have been uploaded to Zenodo at the following link: https://doi.org/10.5281/zenodo.15639948

  • Conflicts of Interest

    The Authors declare no potential conflicts of interest in relation to this study.

  • Authors’ Contributions

    J.W. initiated the study, organized, performed experiments, analyzed data, and wrote the paper; S.C. (Shiyuan Chang) and S.C. (Shuo Chen) performed experiments and analyzed data; Y.Q. designed experiments and analyzed data; W.S. contributed to funding acquisition, analyzed data and wrote the paper. All Authors read and approved the final manuscript.

  • Funding

    This work was supported by the National Natural Science Foundation of China (82003092) and the Natural Science Foundation of Tianjin (23JCZDJC01250).

  • Artificial Intelligence (AI) Disclosure

    During the preparation of this manuscript, a large language model (DeepSeek-R1) was used solely for language editing and stylistic improvements in select paragraphs. No sections involving the generation, analysis, or interpretation of research data were produced by generative AI. All scientific content was created and verified by the authors. Furthermore, no figures or visual data were generated or modified using generative AI or machine learning–based image enhancement tools.

  • Received May 25, 2025.
  • Revision received June 13, 2025.
  • Accepted June 20, 2025.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).

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PPIL1 Drives Hepatocellular Carcinoma Progression and Cancer Stem Cell Self-renewal Through DAAM2-mediated Wnt/β-Catenin Activation
JIAYI WU, SHIYUAN CHANG, SHUO CHEN, YU QI, WEIJUN SU
Cancer Genomics & Proteomics Sep 2025, 22 (5) 760-774; DOI: 10.21873/cgp.20535
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