High expression of CFL1 and phospho-CFL1 predicts poor prognosis and promotes migration of colon cancer cells in vitro

doi:10.12122/j.issn.1673-4254.2026.07.07

Abstract

Abstract:

Objective To investigate the expressions of cofilin-1 (CFL1) and its phosphorylated form p-CFL1 in colon cancer, their relationship with clinicopathological features and patient prognosis, and their regulatory roles in colon cancer cell migration. Methods The expression of CFL1 and p-CFL1 and their correlation with clinicopathological parameters were analyzed using the TCGA database and immunohistochemistry of colon cancer tissue microarrays. Kaplan-Meier survival analysis was used to explore the association of CFL1/p-CFL1 expression levels with survival of colon cancer patients. The localization of CFL1 and p-CFL1 in the nucleus and cytoplasm was observed using immunofluorescence staining. Wild-type (CFL1-WT) and nuclear localization signal mutant-type (CFL1-MUT) overexpression vectors were constructed and transfected into HCT116 cells, and the changes in cell migration were assessed using Transwell chamber migration assay. Transcriptome sequencing combined with KEGG enrichment analysis was performed to identify the differentially expressed genes and signaling pathways. Results Immunohistochemistry results showed significantly increased expressions of CFL1 and p-CFL1 in colon cancer tissues compared with the adjacent tissues (P<0.0001), and their expression levels were positively correlated (r=0.4753, P<0.0001). High expressions of CFL1 and p-CFL1 were significantly correlated with lymph node metastasis, advanced clinical stage, and shorter overall survival of the patients (P<0.05). Immunohistochemistry and immunofluorescence staining demonstrated localization of CFL1 and p-CFL1 in both the cytoplasm and nucleus. Functional experiments showed that overexpression of CFL1-WT and CFL1-MUT both promoted HCT116 cell migration, and CFL1-WT exhibited a stronger effect. KEGG analysis suggested that the genes regulated by CFL1-WT were significantly enriched in tumor metastasis-related pathways involving Ras, PI3K-Akt, and cell adhesion molecules. Conclusion CFL1/p-CFL1 are highly expressed in colon cancer and can be potential biomarkers for poor prognosis. CFL1 overexpression further activates tumor metastasis-related signaling pathways through nuclear localization, thereby promoting colon cancer migration.

Key words: colon cancer, cofilin-1, phosphorylated cofilin-1, nuclear localization, prognosis

Linyu WANG, Xiaoyan WANG, Jijun ZHU, Bo JIANG, Jie XU, Caiyue GAO, Dongdong LI, Yu WANG, Xiaomin LI. High expression of CFL1 and phospho-CFL1 predicts poor prognosis and promotes migration of colon cancer cells in vitro[J]. Journal of Southern Medical University, 2026, 46(7): 1533-1542.

Figures/Tables 10

Fig.1 CFL1 is highly expressed in colon cancer tissues. A: Expression levels of CFL1 in colon adenocarcinoma (COAD) and rectal adenocarcinoma (READ) from the TCGA database. B, C: Representative immunohistochemical staining and the corresponding H-score analysis of CFL1 in colon cancer tissues and paired adjacent normal tissues (n=80). Scale bar=500 µm (upper panel); Scale bar=200 µm (lower panel). *P<0.05, ****P<0.0001.

Fig.2 p-CFL1 is highly expressed in colon cancer tissues. A, B: Representative immunohistochemical staining and the corresponding H-score analysis of p-CFL1 in colon cancer tissues and paired adjacent normal tissues (n=80). Scale bar=500 µm (upper panel); Scale bar=200 µm (lower panel). ****P<0.0001.

Fig.3 Positive correlation between expressions of CFL1 and p-CFL1 in colon cancer tissues. A: Representative images showing consistent expression trends of CFL1 and p-CFL1 in the same colon cancer tissue samples (Scale bar=100 μm). B: Pearson correlation analysis between CFL1 and p-CFL1 expression levels in colon cancer tissues (n=96).

Fig.4 Impact of high expression of CFL1 and p-CFL1 on overall survival of patients with colon cancer. A: Kaplan-Meier survival curves comparing overall survival between patients with high and low expression of CFL1 (P=0.0158). B: Kaplan-Meier survival curves comparing overall survival between patients with high and low expression of p-CFL1 (P=0.0030).

Fig.5 Nuclear localization of CFL1 and p-CFL1 in a subset of colon cancer cases. A: Representative immunohistochemical images showing nuclear and cytoplasmic staining of CFL1 and p-CFL1 in colon cancer tissues (Scale bar=100 μm). B: Immunofluorescence analysis confirms the localization of CFL1 and p-CFL1 in both the nucleus and cytoplasm of HCT116 cells (Scale bar=25 μm).

Fig.6 Overexpression of wild-type and NLS-mutant CFL1 promotes migration of colon cancer cells. A: Sanger sequencing verification showing the presence of the NLS in the CFL1-WT vector and its mutation in the CFL1-MUT vector. B: Western blotting confirms overexpression of CFL1 in HCT116 cells transfected with CFL1-WT and CFL1-MUT vectors. C: Immunofluorescence staining showing that wild-type CFL1 overexpression leads to both nuclear and cytoplasmic distribution, while the NLS-mutant confines CFL1 to the cytoplasm (Scale bar=20 μm). D: Transwell migration assays demonstrating that both CFL1-WT and CFL1-MUT enhancemigration of colon cancer cells (Scale bar=100 μm; n=5). *P<0.05, **P<0.01.

Fig.7 KEGG enrichment analysis of wild-type and NLS-mutant CFL1. A, B: Volcano plots displaying the numbers of differentially expressed genes (DEGs) identified by RNA-seq in HCT116 cells transfected with CFL1-MUT, CFL1-WT, or control vectors. C, D: Bubble plots of KEGG pathway enrichment analysis for up-regulated DEGs (MUT vs NC: 63, WT vs NC: 41) in the CFL1-MUT and CFL1-WT groups, respectively. E, F: Bubble plots of KEGG pathway enrichment analysis for down-regulated DEGs (MUT vs NC: 41, WT vs NC: 54) in the CFL1-MUT and CFL1-WT groups, respectively.

Fig.8 KEGG enrichment analysis of nuclear-localized CFL1. A, B: Venn diagram and heatmaps showing the 31 up-regulated and 45 down-regulated differentially expressed genes in CFL1-WT group compared to the control. C, D: KEGG enrichment analysis of the 31 up-regulated and 45 down-regulated DEGs.

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