CLTC promotes the invasion and metastasis of oral squamous cell carcinoma by activating the ERK signaling pathway

Oral squamous cell carcinoma (OSCC) presents with insidious early symptoms, and most patients have developed lymph node metastasis at the time of initial diagnosis, resulting in poor prognosis and low survival rates. Therefore, exploring novel biomarkers for early diagnosis and targeted therapy strategies is crucial in this field. Clathrin heavy chain (CLTC) is a key regulatory protein in the endocytic process; however, its expression level, clinical significance, and role in invasion and migration in OSCC remain to be elucidated. Revealing these aspects may provide novel targets for OSCC-targeted therapy.

Transcriptomic data and corresponding clinical information from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases were integrated to analyze the expression level of CLTC in OSCC patients. Kaplan-Meier survival analysis was performed to evaluate the impact of CLTC expression on the prognosis of OSCC patients. A nomogram prognostic prediction model was constructed by integrating CLTC expression levels and clinicopathological parameters including patient age, tumor stage, tumor size, and lymph node metastasis status to individually evaluate prognosis in OSCC patients. Gene Set Enrichment Analysis (GSEA) was applied based on Kyoto Encyclopedia of Genes and Genomes (KEGG) database to explore the signaling pathways associated with CLTC. Immunohistochemical assay and Western blotting were employed to detect the protein level of CLTC in OSCC tissues, and analyze its relationship with the clinicopathological features in OSCC patients. Western blotting and RT-qPCR were utilized to measure CLTC expression in HOK, HGE, HSC3, SCC9, CAL27 and SCC25 cell lines. Three small interfering RNAs (siRNAs) were designed according to the CLTC gene sequence. Transfection efficiency was subsequently validated by Western blotting and RT-qPCR. Scratch wound healing assay and Transwell assay were utilized to evaluate the migration and invasion of OSCC cells, respectively. Western blotting was conducted to detect the expression of ERK signaling pathway-related proteins (ERK and pERK).

Bioinformatics analysis showed that CLTC was highly expressed in various cancers, including oral cancer, hepatocellular carcinoma and lung adenocarcinoma, and its high expression was significantly correlated with poor prognosis in patients (P=0.033, HR=1.45, 95%CI: 1.03 to 2.03). The nomogram prediction model for the TCGA cohort showed the AUC values for 1-, 3-, and 5-year survival predictions were 0.660, 0.753, and 0.793, respectively, with calibration curves indicating good consistency between predicted and actual survival probabilities. The nomogram prediction model in the GEO cohort showed the 1-, 3-, and 5-year survival prediction AUC values of 0.722, 0.762, and 0.760, respectively; and calibration curves similarly demonstrated favorable consistency. GSEA results indicated that the differential gene set for CLTC was significantly enriched in multiple cancer-related KEGG pathways. Notably, cancer pathways, Wnt, TGF-β, and MAPK signaling pathways showed significant enrichment effects with high statistical credibility. Clinical specimen detection demonstrated that CLTC expression level in the OSCC tissues was significantly higher than that in paired adjacent non-cancerous tissues (P<0.001), and its expression level was significantly correlated with tumor differentiation degree and lymph node metastasis (P<0.05). CLTC knockdown resulted in markedly inhibited migration and invasion abilities of SCC25 cells (P<0.001) and significantly downregulated levels of ERK signaling pathway-related proteins (P<0.001). Rescue experiment results demonstrated that addition of the ERK signaling pathway activator LM22B-10 partially restored the decreased cell invasion and migration abilities (P<0.01).

CLTC is aberrantly highly expressed in OSCC tissues and cells, and its expression level is significantly correlated with tumor differentiation degree and lymph node metastasis status. CLTC regulates the invasion and migration of OSCC cells, and this effect may be associated with the inhibition of ERK signaling pathway activation.