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Article Open Access

Liquid‑liquid phase separation of PHLDB2 promotes oral squamous cell carcinoma metastasis through regulating epithelial mesenchymal transition and PIK3CA expression

  • Authors:
    • Kunyi Chen
    • Yuhong Wang
    • Jianing Cui
    • Qianrui Liu
    • Xiaoyong Liu
    • Wenjin Wang
    • Jinsong Hou
  • View Affiliations / Copyright

    Affiliations: Department of Oral and Maxillofacial Surgery, Hospital of Stomatology, Sun Yat‑sen University, Guangzhou, Guangdong 510055, P.R. China
    Copyright: © Chen et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 10
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    Published online on: October 22, 2025
       https://doi.org/10.3892/mmr.2025.13720
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Abstract

Liquid‑liquid phase separation (LLPS) contributes to multiple cellular bioprocesses; however, its clinicopathological relevance to oral squamous cell carcinoma (OSCC) remains largely unexplored. In the present study an integrative multi‑omics analysis investigating the prognostic value and molecular functions of LLPS‑related genes (LLPSRGs) in OSCC was conducted by leveraging transcriptomics and clinical data from 302 cases in The Cancer Genome Atlas database and LLPSRGs. A total of two prognostically distinct molecular subtypes were stratified by unsupervised clustering analysis, and a robust prognostic signature comprising seven key LLPSRGs was developed through LASSO regression and multivariate Cox analysis. Functional enrichment analysis highlighted the involvement of this signature in epithelial‑mesenchymal transition (EMT), with PHLDB2 emerging as a core regulator. Notably, the PHLDB2 protein underwent LLPS and formed droplet condensates both outside and in OSCC cells. Moreover, functional experiments revealed that PHLDB2 depletion attenuated the malignant biological behavior of OSCC cells, including cell proliferation, stemness, invasion and migration, and PHLDB2 promoted OSCC progression by regulating the PI3K‑Akt signaling pathway and PIK3CA expression. Retrospective clinical cohort and public dataset analyses validated that high expression of PHLDB2 was significantly associated with lymph node metastasis, higher pathological grade and reduced survival in patients with OSCC. Collectively, the present study established an LLPS‑based prognostic signature for OSCC, and revealed that phase separation of PHLDB2 may drive OSCC progression through regulating EMT and PIK3CA.
View Figures

Figure 1

Workflow diagram of the present
study. DrLLPS, Data Resource of LLPS; LLPS, liquid-liquid phase
separation; LLPSRGs, LLPS-related genes; OSCC, oral squamous cell
carcinoma; TCGA, The Cancer Genome Atlas.

Figure 2

Establishment of two LLPS-related
molecular clusters and construction of a novel prognostic
prediction model based on LLPSRGs for OSCC. (A) Consensus
clustering matrix heatmap illustrating optimal partitioning (k=2)
of TCGA-OSCC samples based on LLPSRG expression patterns. (B)
Expression profile heatmap of differential genes between C1 and C2
clusters. (C) Kaplan-Meier curves for OS of C1 and C2 clusters. (D)
Univariate Cox regression screening identifying seven
prognostically significant LLPSRGs. (E) Construction of a
prognostic prediction model through LASSO coefficient trajectories
with a vertical line indicating optimal λ selection through 10-fold
cross-validation. (F) Expression profile heatmap of the seven model
genes. Training cohort validation including (G) risk stratification
by median cut-off, (H) survival distribution mapping, (I)
Kaplan-Meier curves for OS of the high- and low-risk groups, and
(J) time-dependent ROC analysis demonstrating predictive accuracy
(AUC values for 1, 3 and 5-year survival). Testing cohort
validation including (K) risk stratification by median cutoff, (L)
survival distribution mapping, (M) Kaplan-Meier curves for OS of
the high- and low-risk groups, and (N) time-dependent ROC analysis
demonstrating predictive accuracy (AUC values for 1, 3 and 5-year
survival). (O) Construction of a prognostic nomogram model
integrating risk score with clinical parameters
(Tumor-Node-Metastasis stage, histological grade and age). (P)
Calibration plots showing concordance between predicted and
observed survival probabilities at 1, 3 and 5-year intervals. (Q)
Time-dependent ROC analysis demonstrating predictive accuracy (AUC
values for 1, 3 and 5-year survival). *P<0.05 and ***P<0.001
by multivariate Cox regression analysis. AUC, area under the curve;
LLPS, liquid-liquid phase separation; LLPSRG, LLPS-related gene;
OS, overall survival; OSCC, oral squamous cell carcinoma; ROC,
receiver operating characteristic; TCGA, The Cancer Genome
Atlas.

Figure 3

Functional enrichment analysis and
tumor microenvironment analysis of DEGs between high- and low-risk
groups. (A) DEG analysis identified significant differences between
high- and low-risk groups (threshold: P<0.05 and |log2 fold
change|>1). (B) Minus-vs.-Add plot quality control of the DEG
analysis. (C) Hallmark GSEA revealed enriched pathways in
upregulated DEGs. (D) KEGG GSEA identified significant pathways in
upregulated DEGs (FDR<0.05). (E) GO GSEA determined biological
functions in upregulated DEGs (FDR<0.05). (F) GO GSEA determined
biological functions in downregulated DEGs (FDR<0.05).
*P<0.05, **P<0.01 and ***P<0.001. DEGs, differentially
expressed genes; FDR, false discovery rate; GO, Gene Ontology;
GSEA, gene set enrichment analysis; KEGG, Kyoto Encyclopedia of
Genes and Genomes.

Figure 4

Expression profile of prognostic
model genes in oral squamous cell carcinoma at a single cell level.
(A) Risk scores of cells in primary and metastatic groups in the
GSE234933 dataset based on model gene signatures. (B) Bar plot
showing the distribution of high-risk and low-risk cells in the
primary and metastatic groups, stratified by median risk score. (C)
UMAP visualization of single-cell transcriptomes from the
metastatic group in GSE234933, with cell clusters identified by
unique gene expression profiles. (D) Heatmap showing the expression
of the top three marker genes for each cell cluster. (E) UMAP
visualization of the risk score calculated for the cells from the
metastatic group. (F) UMAP clustering of the tumor cell subtype in
the metastatic group, identified by unique gene expression
profiles. (G) Heatmap showing the expression of the top three
marker genes for each tumor cell subtype. (H) UMAP visualization of
the risk score calculated for the cells of the tumor cell subtype.
(I) Risk scores calculated for the cells of the tumor cell subtype.
(J) Proportion of tumor cell subtypes in the high- and low-risk
groups, stratified by the median risk score. (K) Hallmark GSEA for
pEMT tumor cell subpopulation. **P<0.01 and ***P<0.001. GSEA,
gene set enrichment analysis; NES, normalized enrichment score;
pEMT, partial epithelial-mesenchymal transition; UMAP, Uniform
Manifold Approximation and Projection.

Figure 5

LLPS phenomenon of PHLDB2 in OSCC.
(A) Prediction of disordered protein regions (IUPred2) and
disordered binding motifs (ANCHOR2) in PHLDB2 via the IUPred2A
platform. (B) LLPS propensity scoring of PHLDB2 using PhaSePred.
In vitro droplet assay images of Alexa Fluor 488-labeled
PHLDB2 condensates under increasing (C) protein or (D) NaCl
concentrations (scale bar, 10 µm). (E) EGFP-PHLDB2
plasmid-transfected SCC1 cells showing intracellular droplet
formation (scale bar, 10 µm). (F) FRAP-based quantification of
normalized fluorescence intensity in EGFP-PHLDB2 condensates within
live OSCC cells. (G) FRAP recovery time-lapse imaging of
EGFP-PHLDB2 droplets in live OSCC cells (scale bar, 10 µm). FRAP,
fluorescence recovery after photobleaching; LLPS, liquid-liquid
phase separation; OSCC, oral squamous cell carcinoma.

Figure 6

Promoting effects of PHLDB2 on
proliferation, stemness, invasion and metastasis in OSCC cell
lines. (A) PHLDB2 mRNA levels were decreased in OSCC cells
transfected with PHLDB2-targeting siRNAs (si1/si2). ***P<0.001,
****P<0.0001 vs. control-si. (B) Western blotting confirmed
PHLDB2 protein knockdown following siRNA transfection. OD values
demonstrated changes in (C) HSC3 cells and (D) SCC1 cells upon
PHLDB2 depletion. ***P<0.001. Tumor spheres formed in (E) HSC3
cells and (F) SCC1 cells. Scale bar, 100 µm. (G) Sphere number
decreased in PHLDB2-knockdown HSC3/SCC1 cells. ***P<0.001 vs.
control-si. (H) Sphere diameter decreased in PHLDB2-knockdown
HSC3/SCC1 cells. **P<0.01 vs. control-si. Sphere-forming
capacity decreased in (I) HSC3 cells and (J) SCC1 cells with PHLDB2
knockdown. (K) Representative image of colony formation assays
showed reduction in PHLDB2-knockdown HSC3/SCC1 cells vs. control.
Scale bar, 3 mm. (L) Quantification of colony formation assays
showed reduction in PHLDB2-knockdown HSC3/SCC1 cells.
***P<0.001, ****P<0.0001 vs. control-si. (M) Representative
image of invasion assays indicated suppression in PHLDB2-knockdown
HSC3/SCC1 cells. Scale bar, 50 µm. (N) Quantification of invasion
assays indicated suppression in PHLDB2-knockdown HSC3/SCC1 cells.
***P<0.001 vs. control-si. (O) Representative image of wound
healing assays indicated depletion of migratory capacity in
PHLDB2-knockdown HSC3 cells vs. control. Scale bar, 100 µm. (P)
Wound healing assays indicated depletion of migratory capacity in
PHLDB2-knockdown HSC3 cells. ***P<0.001 vs. control-si. (Q)
Quantification of wound healing assays indicated depletion of
migratory capacity in PHLDB2-knockdown SCC1 cells vs. control.
Scale bar, 100 µm. (R) Wound healing assays indicated depletion of
migratory capacity in PHLDB2-knockdown SCC1 cells. *P<0.05,
**P<0.01 vs. control-si. OSCC, oral squamous cell carcinoma; si,
small interfering.

Figure 7

Exploration of the molecular
mechanisms of PHLDB2 promoting the malignant characteristics of
OSCC. (A) Differential gene analysis identified differentially
expressed genes between PHLDB2 low- and high-expression groups in
TCGA-OSCC cohort. Threshold: P<0.05 and |log2FC|>1. (B)
Differential gene analysis identified differentially expressed
genes between the PHLDB2 knockdown group and the control group.
Thresholds: P<0.05 and |log2FC|>1. (C) Intersection analysis
revealed downregulated pathways shared between PHLDB2-RNAseq and
PHLDB2-TCGA enrichment profiles. (D) Integrated Hallmark pathway
analysis demonstrated enriched pathway for downregulated genes. (E)
Integrated GO pathway analysis identified enriched biological terms
for downregulated genes. (F) Integrated KEGG pathway analysis
showed enriched pathways for downregulated genes. (G) Heatmap
displayed expression patterns of PI3K-Akt signaling pathway genes
in PHLDB2-knockdown vs. control groups. Reverse
transcription-quantitative PCR indicated reduced PIK3CA expression
in PHLDB2-depleted (H) HSC3 and (I) SCC1 cells. **P<0.01,
***P<0.001 vs. control-si. GO, Gene Ontology; GSEA, gene set
enrichment analysis; KEGG, Kyoto Encyclopedia of Genes and Genomes;
NES, normalized enrichment score; OSCC, oral squamous cell
carcinoma; RNAseq, RNA sequencing; TCGA, The Cancer Genome Atlas;
MsigDB, Molecular Signatures Database; si, small interfering.

Figure 8

High expression of PHLDB2 is
associated with a poorer prognosis for patients with OSCC. (A)
Expression levels of PHLDB2 in tumor and normal tissues from
TCGA-OSCC cohort. (B) PHLDB2 expression in paired tumor and normal
samples from TCGA-OSCC cohort. (C) PHLDB2 levels in OSCC tissues
and normal mucosa in the GSE213862 cohort. (D) PHLDB2 expression in
early-stage (T1/T2) and late-stage (T3/T4) tumors from TCGA-OSCC.
(E) Survival comparison between the PHLDB2 high and low expression
groups in TCGA-OSCC. (F and G) IHC staining of PHLDB2 in tumor and
matched normal tissues (Sun Yat-Sen University cohort; scale bar,
50 µm). Semi-quantitative IHC analysis of (H) tumor vs. normal
tissues; (I) LN+ vs. LN− tissues; and (J) G1
vs. G2/G3 tissues. *P<0.05, ***P<0.001 and ****P<0.0001.
CI, confidence interval; IHC, immunohistochemical; LN, lymph node
metastasis; OSCC, oral squamous cell carcinoma; TCGA, The Cancer
Genome Atlas.
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Copy and paste a formatted citation
Spandidos Publications style
Chen K, Wang Y, Cui J, Liu Q, Liu X, Wang W and Hou J: Liquid‑liquid phase separation of PHLDB2 promotes oral squamous cell carcinoma metastasis through regulating epithelial mesenchymal transition and PIK3CA expression. Mol Med Rep 33: 10, 2026.
APA
Chen, K., Wang, Y., Cui, J., Liu, Q., Liu, X., Wang, W., & Hou, J. (2026). Liquid‑liquid phase separation of PHLDB2 promotes oral squamous cell carcinoma metastasis through regulating epithelial mesenchymal transition and PIK3CA expression. Molecular Medicine Reports, 33, 10. https://doi.org/10.3892/mmr.2025.13720
MLA
Chen, K., Wang, Y., Cui, J., Liu, Q., Liu, X., Wang, W., Hou, J."Liquid‑liquid phase separation of PHLDB2 promotes oral squamous cell carcinoma metastasis through regulating epithelial mesenchymal transition and PIK3CA expression". Molecular Medicine Reports 33.1 (2026): 10.
Chicago
Chen, K., Wang, Y., Cui, J., Liu, Q., Liu, X., Wang, W., Hou, J."Liquid‑liquid phase separation of PHLDB2 promotes oral squamous cell carcinoma metastasis through regulating epithelial mesenchymal transition and PIK3CA expression". Molecular Medicine Reports 33, no. 1 (2026): 10. https://doi.org/10.3892/mmr.2025.13720
Copy and paste a formatted citation
x
Spandidos Publications style
Chen K, Wang Y, Cui J, Liu Q, Liu X, Wang W and Hou J: Liquid‑liquid phase separation of PHLDB2 promotes oral squamous cell carcinoma metastasis through regulating epithelial mesenchymal transition and PIK3CA expression. Mol Med Rep 33: 10, 2026.
APA
Chen, K., Wang, Y., Cui, J., Liu, Q., Liu, X., Wang, W., & Hou, J. (2026). Liquid‑liquid phase separation of PHLDB2 promotes oral squamous cell carcinoma metastasis through regulating epithelial mesenchymal transition and PIK3CA expression. Molecular Medicine Reports, 33, 10. https://doi.org/10.3892/mmr.2025.13720
MLA
Chen, K., Wang, Y., Cui, J., Liu, Q., Liu, X., Wang, W., Hou, J."Liquid‑liquid phase separation of PHLDB2 promotes oral squamous cell carcinoma metastasis through regulating epithelial mesenchymal transition and PIK3CA expression". Molecular Medicine Reports 33.1 (2026): 10.
Chicago
Chen, K., Wang, Y., Cui, J., Liu, Q., Liu, X., Wang, W., Hou, J."Liquid‑liquid phase separation of PHLDB2 promotes oral squamous cell carcinoma metastasis through regulating epithelial mesenchymal transition and PIK3CA expression". Molecular Medicine Reports 33, no. 1 (2026): 10. https://doi.org/10.3892/mmr.2025.13720
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